+publisher:"Texas A&M University" +contributor:("Lindahl, Paul")

Texas A&M University

1. Patra, Shachin. Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways.

Degree: PhD, Chemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/173754

► Iron-sulfur (Fe-S) clusters are essential cofactors and are found in all branches of life. In eukaryotes, Fe-S assembly complex is composed of NFS1, ISD11, ACP,… (more)

▼ Iron-sulfur (Fe-S) clusters are essential cofactors and are found in all branches of life. In eukaryotes, Fe-S assembly complex is composed of NFS1, ISD11, ACP, ISCU2, and FXN subunits. NFS1 is a cysteine desulfurase that utilizes a PLP cofactor to extract sulfur from its cysteine substrate and produce a persulfide species on a cysteine residue of a mobile S-transfer loop. Sulfur is then transferred from the mobile S-transfer loop of NFS1 to ISCU2, where it is combined with ferrous iron and electrons to generate [2Fe-2S] clusters. ISD11 and ACP are critical for the stability of the Fe-S assembly complex and may modulate NFS1 function. Eukaryotic ISC pathway is characterized by low activity of SDA and significant rate enhancement by FXN in presence of ISCU2. Prokaryotic cysteine desulfurase IscS (59% identity) stable without accessory proteins and highly active. Interestingly, both FXN and homolog CyaY activates/inhibits the eukaryotic/prokaryotic Fe-S assembly respectively. Here, we investigated the role of FXN/CyaY in each step of Fe-S biosynthesis. We found that S-transfer loop cysteine participates in cys-aldimine formation, cys-quinonoid decay, persulfide formation on itself, and transfer of this sulfane sulfur to scaffold protein. Interestingly, in eukaryotes, FXN enhances all these steps but not as fast as IscS. All these data support a model in which monomeric cysteine desulfurase architecture (such as in SDAvec) promotes non-productive conformations of S-transfer loop of cysteine desulfurase resulting in low activity. FXN binding (in eukaryotic system) excludes non-productive conformations and directs the trajectory of the NFS1 mobile S-transfer loop to position the cysteine to function as a general acid, nucleophile, and sulfur delivery agent in different steps and accelerates Fe-S cluster biosynthesis. In prokaryotes, because of the closed architecture, the other subunit of cysteine desulfurase regulates S-transfer loop trajectory (better than the FXN in eukaryotic system) and therefore have high activity even in absence of FXN. Consistent with this, IscS^S10Q variant exhibits significantly weaker dimer interface, and concentration dependent enhancement of dimer concentration and activity. Also, data indicate that reduced FDX2 is probably not the electron source but sensitizer of persulfide on scaffold protein towards reduction by reduced glutathione. Advisors/Committee Members: Barondeau, David (advisor), Begley, Tadhg (committee member), Raushel, Frank (committee member), Lindahl, Paul (committee member).

Subjects/Keywords: Iron Sulfur Cluster; Cysteine Desulfurase; Frataxin; Friedreich's Ataxia

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APA (6^th Edition):

Patra, S. (2018). Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/173754

Chicago Manual of Style (16^th Edition):

Patra, Shachin. “Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways.” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/173754.

MLA Handbook (7^th Edition):

Patra, Shachin. “Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways.” 2018. Web. 28 Feb 2021.

Vancouver:

Patra S. Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways. [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/173754.

Council of Science Editors:

Patra S. Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways. [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/173754

Texas A&M University

2. Wang, Kecheng. Synthesis of Porphyrinic Metal Organic Frameworks with High Robustness and Catalytic Activity.

Degree: PhD, Chemistry, 2017, Texas A&M University

URL: http://hdl.handle.net/1969.1/166070

► MOFs are ideal platforms to immobilize porphyrins and their derivatives. MOF’s high surface areas and rigid structures not only make porphyrin moieties approachable substrates but… (more)

▼ MOFs are ideal platforms to immobilize porphyrins and their derivatives. MOF’s high surface areas and rigid structures not only make porphyrin moieties approachable substrates but also prevent the dimerization of their active centers. These advantages effectively enhance the reactivity and optical performance of porphyrins. Extensive study has been done to synthesize porphyrinic MOFs, however, many of the previously reported porphyrinic MOFs suffer from weak chemical stabilities, which severely hinders their wide utilization. My study is focused on the design and syntheses of porphyrinic MOFs with high robustness in various chemical environments. Three porphyrinic MOFs with excellent chemical stability, namely PCN-600, PCN-601 and PCN-602, were obtained, and their performances as catalysts in different reactions were explored. PCN-600, a Fe-based mesoporous porphyrinic MOF was successfully synthesized through rational topological design and KTDA method. It exhibits high chemical stability in aqueous solutions with pH values ranging from 2 to 11. The catalytic activity of PCN-600(Fe) (with Fe³⁺ at the porphyrin center) was confirmed by its excellent performance in the co-oxidation of phenol and 4-aminoantipyrine (4-AAP) by H₂O₂. Most of the reported stable porphyrinic MOFs, including PCN-22Xs (X = 2, 3, 4, 5) and PCN-600, are constructed by high-valent metal ions and carboxylate-based porphyrinic ligands. Though these materials have very high robustness in acidic solutions, they are relatively vulnerable in aqueous solutions containing some coordinating anions, such as OH⁻, F⁻, CO₃²⁻ and PO₄³⁻. To address these problems, two iso-structural porphyrinic MOFs constructed by pyrazolate-terminated ligands were synthesized, namely PCN-601 and PCN-602. These materials exhibit extraordinary stability in the solutions of NaOH, KF, Na₂CO₃ and K₃PO₄. Their chemical stabilities were rationalized from both thermodynamic and kinetic perspectives. The catalytic performance of PCN-601(Mn) and PCN-602(Mn) (with Mn³⁺ at the coordination centers of the porphyrinic ligands) in C-H halogenation reactions was explored. Compared with PCN-601(Mn), PCN-602(Mn) has a higher porosity and displays a better activity as a recyclable hetergeneous catalyst. Advisors/Committee Members: Zhou, Hongcai (advisor), Dunbar, Kim (committee member), Lindahl, Paul (committee member), Jeong, Hae-Kwon (committee member).

Subjects/Keywords: MOFs; Porphyrin; Stability; Catalysts

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wang, K. (2017). Synthesis of Porphyrinic Metal Organic Frameworks with High Robustness and Catalytic Activity. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/166070

Chicago Manual of Style (16^th Edition):

Wang, Kecheng. “Synthesis of Porphyrinic Metal Organic Frameworks with High Robustness and Catalytic Activity.” 2017. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/166070.

MLA Handbook (7^th Edition):

Wang, Kecheng. “Synthesis of Porphyrinic Metal Organic Frameworks with High Robustness and Catalytic Activity.” 2017. Web. 28 Feb 2021.

Vancouver:

Wang K. Synthesis of Porphyrinic Metal Organic Frameworks with High Robustness and Catalytic Activity. [Internet] [Doctoral dissertation]. Texas A&M University; 2017. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/166070.

Council of Science Editors:

Wang K. Synthesis of Porphyrinic Metal Organic Frameworks with High Robustness and Catalytic Activity. [Doctoral Dissertation]. Texas A&M University; 2017. Available from: http://hdl.handle.net/1969.1/166070

Texas A&M University

3. Patra, Shachin. Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways.

Degree: PhD, Chemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/173849

► Iron-sulfur (Fe-S) clusters are essential cofactors and are found in all branches of life. In eukaryotes, Fe-S assembly complex is composed of NFS1, ISD11, ACP,… (more)

▼ Iron-sulfur (Fe-S) clusters are essential cofactors and are found in all branches of life. In eukaryotes, Fe-S assembly complex is composed of NFS1, ISD11, ACP, ISCU2, and FXN subunits. NFS1 is a cysteine desulfurase that utilizes a PLP cofactor to extract sulfur from its cysteine substrate and produce a persulfide species on a cysteine residue of a mobile S-transfer loop. Sulfur is then transferred from the mobile S-transfer loop of NFS1 to ISCU2, where it is combined with ferrous iron and electrons to generate [2Fe-2S] clusters. ISD11 and ACP are critical for the stability of the Fe-S assembly complex and may modulate NFS1 function. Eukaryotic ISC pathway is characterized by low activity of SDA and significant rate enhancement by FXN in presence of ISCU2. Prokaryotic cysteine desulfurase IscS (59% identity) stable without accessory proteins and highly active. Interestingly, both FXN and homolog CyaY activates/inhibits the eukaryotic/prokaryotic Fe-S assembly respectively. Here, we investigated the role of FXN/CyaY in each step of Fe-S biosynthesis. We found that S-transfer loop cysteine participates in cys-aldimine formation, cys-quinonoid decay, persulfide formation on itself, and transfer of this sulfane sulfur to scaffold protein. Interestingly, in eukaryotes, FXN enhances all these steps but not as fast as IscS. All these data support a model in which monomeric cysteine desulfurase architecture (such as in SDAvec) promotes non-productive conformations of S-transfer loop of cysteine desulfurase resulting in low activity. FXN binding (in eukaryotic system) excludes non-productive conformations and directs the trajectory of the NFS1 mobile S-transfer loop to position the cysteine to function as a general acid, nucleophile, and sulfur delivery agent in different steps and accelerates Fe-S cluster biosynthesis. In prokaryotes, because of the closed architecture, the other subunit of cysteine desulfurase regulates S-transfer loop trajectory (better than the FXN in eukaryotic system) and therefore have high activity even in absence of FXN. Consistent with this, IscS^S10Q variant exhibits significantly weaker dimer interface, and concentration dependent enhancement of dimer concentration and activity. Also, data indicate that reduced FDX2 is probably not the electron source but sensitizer of persulfide on scaffold protein towards reduction by reduced glutathione. Advisors/Committee Members: Barondeau, David (advisor), Begley, Tadhg (committee member), Raushel, Frank (committee member), Lindahl, Paul (committee member).

Subjects/Keywords: Iron Sulfur Cluster; Cysteine Desulfurase; Frataxin; Friedreich's Ataxia

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Patra, S. (2018). Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/173849

Chicago Manual of Style (16^th Edition):

Patra, Shachin. “Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways.” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/173849.

MLA Handbook (7^th Edition):

Patra, Shachin. “Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways.” 2018. Web. 28 Feb 2021.

Vancouver:

Patra S. Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways. [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/173849.

Council of Science Editors:

Patra S. Mechanistic Analysis of the Role of Frataxin in Bacterial and Eukaryotic Fe-S Cluster Biosynthetic Pathways. [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/173849

Texas A&M University

4. Das, Deepika. Mechanistic Analysis of Cluster Transfer to Apo-acceptors in ISC Pathway.

Degree: PhD, Chemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/173609

► Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for some of the most important reactions in nature, including nitrogen fixation, oxidative phosphorylation, and… (more)

▼ Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for some of the most important reactions in nature, including nitrogen fixation, oxidative phosphorylation, and photosynthesis. Multiple biosynthetic pathways have evolved for assembling Fe-S clusters and delivering them to protein targets. The ISC (Iron-Sulfur Cluster assembly) pathway is found in many prokaryotes and in eukaryotic mitochondria. A central component of the pathway is a cysteine desulfurase, which uses a pyridoxal phosphate (PLP) cofactor to convert cysteine to alanine and functions as a sulfur source. The scaffold protein IscU combines this sulfur with ferrous iron and electrons to synthesize Fe-S clusters. The Fe-S clusters can then be transferred to acceptor proteins such as the monothiol glutaredoxins (Grx4 in bacteria, GRX5 in humans) and ferredoxin (Fdx). The chaperone co-chaperone pair (HscA-HscB) accelerate cluster transfer reactions from holo-IscU to Grx4 and Fdx. In eukaryotes, Fe-S cluster biosynthesis is stimulated by frataxin (FXN), which is associated with the neurodegenerative disease Friedreich’s ataxia (FRDA). To gain a mechanistic understanding into these processes, we employed stopped-flow kinetic analysis and developed visible circular dichroism, fluorescence reporter, and radioactivity-based assays. Importantly, we develop the first complete functional assay in which Fe-S clusters are built and then transferred to a target protein. Here, studies will be presented that show (1) FXN accelerates specific steps in the PLP-associated chemistry of the cysteine desulfurase, interprotein sulfur transfer, and intermediate Fe-S cluster formation on the scaffold protein; (2) a FXN suppressor mutant identified on the scaffold protein functions differently than FXN, accelerating cluster transfer from the scaffold protein to GRX5; (3) glutaredoxin functions as an intermediate carrier in cluster transfer reactions from IscU to apo acceptor proteins Fdx and HcaC; and (4) chaperones accelerate cluster transfer reactions from IscU to Grx4 and do not appear to assist in the folding or unfolding of the target proteins. Overall, this work provides new mechanistic insight into specific steps of Fe-S cluster assembly and transfer and may provide new opportunities for drug discovery to treat FRDA. Advisors/Committee Members: Barondeau, David P (advisor), Raushel, Frank (committee member), Lindahl, Paul (committee member), Rye, Hays (committee member).

Subjects/Keywords: Iron-sulfur clusters; ISC pathway; Cluster transfer; Fe-S cluster biosynthesis

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Das, D. (2018). Mechanistic Analysis of Cluster Transfer to Apo-acceptors in ISC Pathway. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/173609

Chicago Manual of Style (16^th Edition):

Das, Deepika. “Mechanistic Analysis of Cluster Transfer to Apo-acceptors in ISC Pathway.” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/173609.

MLA Handbook (7^th Edition):

Das, Deepika. “Mechanistic Analysis of Cluster Transfer to Apo-acceptors in ISC Pathway.” 2018. Web. 28 Feb 2021.

Vancouver:

Das D. Mechanistic Analysis of Cluster Transfer to Apo-acceptors in ISC Pathway. [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/173609.

Council of Science Editors:

Das D. Mechanistic Analysis of Cluster Transfer to Apo-acceptors in ISC Pathway. [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/173609

Texas A&M University

5. Cockrell, Allison Leigh. Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae.

Degree: PhD, Biochemistry, 2013, Texas A&M University

URL: http://hdl.handle.net/1969.1/151772

► Transition metals play essential roles in biological systems, but Fe can also be toxic to cells. In order to maintain this balance between necessity and… (more)

▼ Transition metals play essential roles in biological systems, but Fe can also be toxic to cells. In order to maintain this balance between necessity and toxicity mechanisms are employed for regulating and storing intracellular Fe. In Saccharomyces cerevisiae, vacuoles are responsible for sequestering, storing, and supplying Fe to the cytosol. Many of the proteins and regulatory pathways involved in Fe trafficking and storage in S. cerevisiae have been identified, but the forms of Fe which are involved in these processes have not been fully characterized. In these studies, biophysical and bioanalytical techniques were used to study intracellular Fe distributions in S. cerevisiae cells and organelles. Ultimately, Fe-containing species were biophysically characterized and absolute Fe concentrations in cells and organelles were quantified. The motivation for these studies stemmed from previous studies which revealed that the majority of the whole-cell Fe is a non-heme, high-spin (NHHS) form of Fe^(3+). This Fe is not localized to the mitochondria. The purpose of these studies was to determine if the vacuoles contained this NHHS Fe^(3+). A large-scale isolation procedure was developed to obtain purified vacuoles from S. cerevisiae and to investigate the Fe in these organelles. Mössbauer and EPR analysis revealed that the primary form of Fe in vacuoles is a mononuclear, NHHS Fe^(3+) species. A second form of Fe was also observed as superparamagnetic ferric phosphate nanoparticles (NP). By investigating model compounds of Fe and polyphosphate we determined that a shift in vacuolar pH induces the conversion between NHHS Fe^(3+) and NP. These results showed that there are at least two forms of Fe in vacuoles, and that the ratio of these two forms is dependent upon the pH of these organelles. Biophysical analyses of whole cells also revealed the presence of low concentrations of a non-heme, high-spin Fe^(2+) species. The goal of these next projects was to determine if this NHHS Fe^(2+) species was localized to the cytosol. Genetic strains lacking or over-expressing the vacuolar Fe import protein Ccc1p were studied by Mössbauer spectroscopy (∆CCC1 and CCC1-up, respectively). ∆CCC1 cells showed low vacuolar Fe (NHHS Fe3+ and NP), and increased NHHS Fe^(2+). We hypothesize that this NHHS Fe^(2+) is cytosolic Fe. We also propose that this NHHS Fe^(2+) is involved in the regulating intracellular Fe levels. CCC1-up cells accumulated more Fe than wild-type (WT) cells, and showed elevated levels of vacuolar Fe (NHHS Fe^(3+) and NP). These cells also accumulated high levels of NHHS Fe^(2+). The CCC1-up cells exhibited an adenine deficient phenotype, where the cells developed a red color during growth. With excess adenine the levels of NHHS Fe^(2+) declined, which indicated that this Fe accumulation was related to adenine deficiency. We conclude that adenine deficiency leads to the accumulation of a sequestered (possibly vacuolar) form of NHHS Fe^(2+). Overall, we have identified two separate pools of NHHS Fe^(2+) in ∆CCC1 and… Advisors/Committee Members: Lindahl, Paul A (advisor), Raushel, Frank (committee member), Igumenova, Tatyana (committee member), Kaplan, Craig (committee member).

Subjects/Keywords: Iron; Yeast; Vacuoles; Spectroscopy; Mossbauer; EPR; CCC1; Cytosol; Mitochondria

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Cockrell, A. L. (2013). Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/151772

Chicago Manual of Style (16^th Edition):

Cockrell, Allison Leigh. “Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae.” 2013. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/151772.

MLA Handbook (7^th Edition):

Cockrell, Allison Leigh. “Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae.” 2013. Web. 28 Feb 2021.

Vancouver:

Cockrell AL. Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae. [Internet] [Doctoral dissertation]. Texas A&M University; 2013. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/151772.

Council of Science Editors:

Cockrell AL. Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae. [Doctoral Dissertation]. Texas A&M University; 2013. Available from: http://hdl.handle.net/1969.1/151772

Texas A&M University

6. Wang, Xiaoshan. The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery.

Degree: PhD, Chemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/173491

► This study is composed of two parts. In the first part, we discussed nitrilimine-alkene cycloaddition for protein labeling. The mechanism of this nitrilimine-alkene cycloaddition was… (more)

▼ This study is composed of two parts. In the first part, we discussed nitrilimine-alkene cycloaddition for protein labeling. The mechanism of this nitrilimine-alkene cycloaddition was proposed, and thereby the best experimental condition for this protein labeling approach was investigated. The transient formation of nitrilimine in aqueous conditions is greatly influenced by pH and chloride. In basic conditions (pH 10) with no chloride, a diarylnitrilimine precursor readily ionizes to form diarylnitrilimine that reacts almost instantly with an acrylamide-containing protein and fluorescently labels it. In the second part, a novel method for the synthesis of phage-displayed cyclic peptide libraries is presented. Cyclic peptide drugs are appealing in the drug discovery research area due to their unique advantages including high affinity, high specificity, low toxicity, and high cellular and serum stability. In order to identify cyclic peptides as therapeutic agents, during my graduate study I have developed a phage display-based methodology that integrates the genetic noncanonical amino acid (ncAA) mutagenesis technique for the synthesis of novel phage-displayed cyclic peptides through simultaneous 1,4-addition between a cysteine thiol group and acrylamide moiety in Nε -acryloyl-lysine (AcrK), a ncAA. Both cysteine and AcrK are genetically coded. The success of using a cysteine and an AcrK to cyclic a peptide in a model protein and phages was validated by various approaches. In order to afford a library, a phage-displayed cyclic peptide library was constructed by inserting a consecutive but sequence-randomized 6-mer peptide flanked by an amino side cysteine and a carboxyl side AcrK. Panning of the synthesized phage-displayed cyclic peptide library was performed against two target proteins that are tobacco etch virus (TEV) protease and histone deacetylase 8 (HDAC8). A lot of high-affinity phage clones were isolated and collected. DNA sequencing of these selected clones led to the identification of several peptides that potentially inhibit TEV protease and HDAC8. To confirm their potencies as inhibitors, abundant peptides and their fluorophore-conjugated derivatives were synthesized through solid-phase peptide synthesis (SPPS). Measurements of fluorescence polarization change and IC50 value of these peptides when they bound to TEV protease and HDAC8 were performed. Overall, we have mechanistically characterized the nitrilimine-alkene cycloaddition reaction and developed a novel approach for the synthesis of phage-displayed cyclic peptide libraries. The selection of displayed peptides against TEV protease and HDAC8 has resulted in multiple peptides that display high potencies against these two enzymes. Advisors/Committee Members: Liu, Wenshe (advisor), Begley, Tadhg (committee member), Lindahl, Paul (committee member), Raushel, Frank (committee member).

Subjects/Keywords: nitrilimine-alkene cycloaddition; cyclic peptide; HDAC8 inhibitor

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wang, X. (2018). The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/173491

Chicago Manual of Style (16^th Edition):

Wang, Xiaoshan. “The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery.” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/173491.

MLA Handbook (7^th Edition):

Wang, Xiaoshan. “The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery.” 2018. Web. 28 Feb 2021.

Vancouver:

Wang X. The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery. [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/173491.

Council of Science Editors:

Wang X. The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery. [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/173491

Texas A&M University

7. Fox, Nicholas G. A Biophysical Approach to Investigate the Human Fe-S Cluster Assembly Pathway.

Degree: PhD, Chemistry, 2014, Texas A&M University

URL: http://hdl.handle.net/1969.1/153303

► Iron sulfur (Fe-S) clusters are essential cofactors that function in electron transport, catalyzing substrate turnover, environmental sensing, and initiating radical chemistry. Elaborate multi-component systems have… (more)

▼ Iron sulfur (Fe-S) clusters are essential cofactors that function in electron transport, catalyzing substrate turnover, environmental sensing, and initiating radical chemistry. Elaborate multi-component systems have evolved to protect organisms from the toxic effects of free iron and sulfide ions while promoting the efficient biosynthesis of these cofactors. The in vivo loss of frataxin (FXN) function results in depleted activity of Fe-S enzymes and is directly linked to the fatal and incurable neurodegenerative disease Friedreich’s ataxia (FRDA). Previously, our lab discovered the cysteine desulfurase and Fe-S assembly activities of the human Fe-S assembly complex (SDU), which consists of the cysteine desulfurase complex NFS1-ISD11 and scaffold protein ISCU2, are greatly stimulated by FXN binding and forming the SDUF complex. This dissertation’s objectives were to identify critical FXN interactions for binding and activation of the SDU complex, investigate the interprotein sulfur transfer reaction between NFS1 and ISCU2, and provide mechanistic details of Fe-S assembly on the SDUF complex. First, surface residues on FXN were substituted with alanine or glycine and the ability of each variant to bind and activate the SDU complex was assessed. These experiments revealed a localized “hotspot” of critical residues on FXN, which could aid in designing small peptide mimics for FRDA therapeutics. Second, ^(35)S-radiolabeling experiments indicated FXN accelerates the accumulation of persulfide species on NFS1 and ISCU2. The ISCU2 persulfide species was established as a viable intermediate in Fe-S cluster biosynthesis by tracking the ^(35)S-radiolabel as it converts from a persulfide species to a [2Fe-2S] cluster. Additional mutagenic, enzymatic, and spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN-based activation, whereas C35, C61, and C104, are all essential for Fe-S cluster biosynthesis. These results lead to an activation model in which FXN facilitates sulfur transfer from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis and favors helix-to-coil interconversion on ISCU2. Third, UV-visible, circular dichroism, and Mössbauer spectroscopic studies indicated the SDUF complex synthesizes transient [2Fe-2S] clusters that readily transfer to thiol-containing acceptor molecules. Moreover, these studies revealed competing DTT-mediated transfer and mineralization chemistry that cause complications when studying the mechanism of Fe-S cluster biosynthesis. Advisors/Committee Members: Barondeau, David P (advisor), Lindahl, Paul (committee member), Sacchettini, James (committee member), Liu, Wenshe (committee member).

Subjects/Keywords: Fe-S

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Fox, N. G. (2014). A Biophysical Approach to Investigate the Human Fe-S Cluster Assembly Pathway. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/153303

Chicago Manual of Style (16^th Edition):

Fox, Nicholas G. “A Biophysical Approach to Investigate the Human Fe-S Cluster Assembly Pathway.” 2014. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/153303.

MLA Handbook (7^th Edition):

Fox, Nicholas G. “A Biophysical Approach to Investigate the Human Fe-S Cluster Assembly Pathway.” 2014. Web. 28 Feb 2021.

Vancouver:

Fox NG. A Biophysical Approach to Investigate the Human Fe-S Cluster Assembly Pathway. [Internet] [Doctoral dissertation]. Texas A&M University; 2014. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/153303.

Council of Science Editors:

Fox NG. A Biophysical Approach to Investigate the Human Fe-S Cluster Assembly Pathway. [Doctoral Dissertation]. Texas A&M University; 2014. Available from: http://hdl.handle.net/1969.1/153303

Texas A&M University

8. Tripathi, Utkarsh. PARTIAL INHIBITION OF MITOCHONDRIAL COMPLEX I ACTIVATES STRESS RESPONSE PATHWAYS INDUCING A PROTECTION AGAINST OXIDATIVE STRESS.

Degree: MS, Biochemistry, 2017, Texas A&M University

URL: http://hdl.handle.net/1969.1/187299

► We have previously shown that partial inhibition of mitochondrial complex I activity with a small molecule tricycle pyrone compound (code name CP2) averted the development… (more)

▼ We have previously shown that partial inhibition of mitochondrial complex I activity with a small molecule tricycle pyrone compound (code name CP2) averted the development of cognitive and behavior phenotypes in multiple transgenic mouse models of Alzheimer’s disease (AD). One of the beneficial effects of CP2 involved the protection against oxidative stress in primary mouse neurons, human cells and in mice fed with a high fat diet. In the current study, I investigated the molecular mechanism of CP2-induced neuroprotection and established the translational potential of this therapeutics using murine, human and Drosophila model systems. Target identification revealed that CP2 competes with a flavin mononucleotide (FMN) for the binding to the redox subunit of mitochondrial complex I. Bioenergetics studies showed that CP2 mildly inhibits basal cellular respiration simultaneously leading to a dose-dependent generation of a sub-lethal level of reactive oxygen species (ROS). This increase in ROS rapidly activates cytosolic signaling pathways including the nuclear factor erythroid 2-related factor 2/antioxidant response element (NRF2/ARE) activation to induce a protection against oxidative stress. This retrograde response is known as mitohormesis, and the genetic validation of this phenomenon was done in the longevity studies in several model organisms including Drosophila and C. elegans. Mitohormetic nature of CP2-induced protection against oxidative stress was confirmed using pharmacological and genetic manipulations in human cells, in Drosophila, and in the ARE reporter mice in vivo, and validated in mouse models of the AD. CP2 treatment also reduced the extent of oxidative damage and enhanced the survival in wild-type (WT) mice fed with a high fat diet (HFD), which could be attributed to mitohormetic induction of the NRF2/ARE pathway. In summary, this study provides compelling evidence that mitohormetic activation of the NRF2/ARE pathway using small molecule partial inhibitor of mitochondrial complex I could be beneficial in multiple human conditions where oxidative stress contributes to the disease phenotype. Advisors/Committee Members: Lindahl, Paul (advisor), Bryk, Mary (committee member), Chapkin, Robert (committee member), Trushina, Eugenia (committee member).

Subjects/Keywords: mitohormesis; mitochondrial complex I inhibitor; Alzheimer's Disease; oxidative stress

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APA (6^th Edition):

Tripathi, U. (2017). PARTIAL INHIBITION OF MITOCHONDRIAL COMPLEX I ACTIVATES STRESS RESPONSE PATHWAYS INDUCING A PROTECTION AGAINST OXIDATIVE STRESS. (Masters Thesis). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/187299

Chicago Manual of Style (16^th Edition):

Tripathi, Utkarsh. “PARTIAL INHIBITION OF MITOCHONDRIAL COMPLEX I ACTIVATES STRESS RESPONSE PATHWAYS INDUCING A PROTECTION AGAINST OXIDATIVE STRESS.” 2017. Masters Thesis, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/187299.

MLA Handbook (7^th Edition):

Tripathi, Utkarsh. “PARTIAL INHIBITION OF MITOCHONDRIAL COMPLEX I ACTIVATES STRESS RESPONSE PATHWAYS INDUCING A PROTECTION AGAINST OXIDATIVE STRESS.” 2017. Web. 28 Feb 2021.

Vancouver:

Tripathi U. PARTIAL INHIBITION OF MITOCHONDRIAL COMPLEX I ACTIVATES STRESS RESPONSE PATHWAYS INDUCING A PROTECTION AGAINST OXIDATIVE STRESS. [Internet] [Masters thesis]. Texas A&M University; 2017. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/187299.

Council of Science Editors:

Tripathi U. PARTIAL INHIBITION OF MITOCHONDRIAL COMPLEX I ACTIVATES STRESS RESPONSE PATHWAYS INDUCING A PROTECTION AGAINST OXIDATIVE STRESS. [Masters Thesis]. Texas A&M University; 2017. Available from: http://hdl.handle.net/1969.1/187299

Texas A&M University

9. Sun, Yujia. Construction of Highly Stable Metal-Organic Frameworks with Multiple Functionalities.

Degree: PhD, Chemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/174404

► Metal-organic frameworks (MOFs) are a class of newly emerged crystalline porous materials consisting of metal ions or clusters and organic linkers. Through judicious choice of… (more)

▼ Metal-organic frameworks (MOFs) are a class of newly emerged crystalline porous materials consisting of metal ions or clusters and organic linkers. Through judicious choice of inorganic joints and organic struts, the structure, porosity and functionality of MOFs can be tuned. However, the lack of high stability of most of the reported MOFs as well as limited methods to introduce multiple functionalities into the framework hinders the exploration of MOFs towards a wide variety of potential applications. The main goal of this research is to develop synthetic strategies to construct MOFs with high stability and multiple functionalities. Firstly, a brief introduction of MOFs was provided, focusing on strategies to increase their stability and introduce functionalities. Secondly, a facile one-pot synthetic strategy was developed to introduce porphyrin into highly stable UiO-66 homogeneously. The crystal structure, morphology, and ultrahigh chemical stability of UiO-66 were well maintained in the functionalized MOFs. In addition, the amount of integrated porphyrin can be gradually tuned. Thirdly, a general in situ secondary ligand incorporation (ISLI) strategy was investigated to synthesize multivariate UiO series of MOFs. Both experimental and computational studies were carried out to understand the chemistry behind this strategy. Fourthly, ISLI strategy was further applied in highly stable Zr-MOFs constructed from multitopic ligands to incorporate multiple functionalities. Fifthly, a porphyrin and pyrene-based mixed-ligand MOF with high stability and novel topology was synthesized. This MOF provides an ideal platform for further functionalization and exploration of new structures. In summary, different strategies were investigated to construct highly stable MOFs with the incorporation of multiple functionalities. These studies provide useful tools to explore stable MOFs with desired multifunctionality for potential applications. Advisors/Committee Members: Zhou, Hongcai (advisor), Clearfield, Abraham (advisor), Lindahl, Paul (committee member), Grunlan, Jaime (committee member).

Subjects/Keywords: Metal-Organic Frameworks; Porphyrin; Multifunctionality

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APA (6^th Edition):

Sun, Y. (2018). Construction of Highly Stable Metal-Organic Frameworks with Multiple Functionalities. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/174404

Chicago Manual of Style (16^th Edition):

Sun, Yujia. “Construction of Highly Stable Metal-Organic Frameworks with Multiple Functionalities.” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/174404.

MLA Handbook (7^th Edition):

Sun, Yujia. “Construction of Highly Stable Metal-Organic Frameworks with Multiple Functionalities.” 2018. Web. 28 Feb 2021.

Vancouver:

Sun Y. Construction of Highly Stable Metal-Organic Frameworks with Multiple Functionalities. [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/174404.

Council of Science Editors:

Sun Y. Construction of Highly Stable Metal-Organic Frameworks with Multiple Functionalities. [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/174404

Texas A&M University

10. Wofford, Joshua D. Use of Mathematical Modeling and Other Biophysical Methods for Insights into Iron-Related Phenomena of Biological Systems.

Degree: PhD, Chemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/174397

► Iron is a crucial nutrient in most living systems. It forms the active centers of many proteins that are critical for many cellular functions, either… (more)

▼ Iron is a crucial nutrient in most living systems. It forms the active centers of many proteins that are critical for many cellular functions, either by themselves or as Fe-S clusters and hemes. However, Fe is potentially toxic to the cell in high concentrations and must be tightly regulated. There has been much work into understanding various pieces of Fe trafficking and regulation, but integrating all of this information into a coherent model has proven difficult. Past research has focused on different Fe species, including cytosolic labile Fe or mitochondrial Fe-S clusters, as being the main regulator of Fe trafficking in yeast. Our initial modeling efforts demonstrate that both cytosolic Fe and mitochondrial ISC assembly are required for proper regulation. More recent modeling efforts involved a more rigorous multi tiered approach. Model simulations were optimized against experimental results involving respiring wild-type and Mrs3/4-deleted yeast. Simulations from both modeling studies suggest that mitochondria possess a “respiratory shield” that prevents a vicious cycle of nanoparticle formation, ISC loss, and subsequent loading of mitochondria with iron. Work has also been done in understanding an accumulation of Fe in stationary grown yeast cells. This accumulated Fe was found to be localized to the cell wall, and can be used as cells are metabolically reactivating by being placed into fresh media. A mathematical model has been developed to describe the metabolism of oxygen and nutrients in the autocatalytic production of active cells, with subsequent deactivation of cells as nutrients became limiting. E. coli have similar Fe contents relative to mitochondria, and they also appear to also employ a “respiratory shield”. This hypothesis was tested by either inhibiting respiratory complexes with CN, or by growing cells into a metabolically inactive stationary growth state. The generated nanoparticles were not associated with ferritins, which is surprising given that much of the literature claims that ferritin Fe makes up a large portion of cellular Fe. The iron content of murine hearts was also studied. Previous work from the Lindahl lab focused on murine brains and livers, which contain ferritin at young and old ages, while losing it in middle age. Hearts differ from these two organs, in that they mainly contain respiratory iron sulfur clusters, and only gain ferritin as the mice approach old age. Advisors/Committee Members: Lindahl, Paul (advisor), North, Simon (committee member), Barondeau, David (committee member), Gohil, Vishal (committee member).

Subjects/Keywords: Chemistry; Iron; Biology; Trafficking; Regulation; Modeling

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APA (6^th Edition):

Wofford, J. D. (2018). Use of Mathematical Modeling and Other Biophysical Methods for Insights into Iron-Related Phenomena of Biological Systems. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/174397

Chicago Manual of Style (16^th Edition):

Wofford, Joshua D. “Use of Mathematical Modeling and Other Biophysical Methods for Insights into Iron-Related Phenomena of Biological Systems.” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/174397.

MLA Handbook (7^th Edition):

Wofford, Joshua D. “Use of Mathematical Modeling and Other Biophysical Methods for Insights into Iron-Related Phenomena of Biological Systems.” 2018. Web. 28 Feb 2021.

Vancouver:

Wofford JD. Use of Mathematical Modeling and Other Biophysical Methods for Insights into Iron-Related Phenomena of Biological Systems. [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/174397.

Council of Science Editors:

Wofford JD. Use of Mathematical Modeling and Other Biophysical Methods for Insights into Iron-Related Phenomena of Biological Systems. [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/174397

Texas A&M University

11. Jhurry, Nema. Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae.

Degree: PhD, Biochemistry, 2013, Texas A&M University

URL: http://hdl.handle.net/1969.1/151675

► The speciation of iron in intact Jurkat cells and their isolated mitochondria was assessed using biophysical methods. [Fe4S4]^(2+) clusters, low-spin (LS) Fe^(II) heme centers, non-heme… (more)

▼ The speciation of iron in intact Jurkat cells and their isolated mitochondria was assessed using biophysical methods. [Fe4S4]^(2+) clusters, low-spin (LS) Fe^(II) heme centers, non-heme high-spin (NHHS) FeII species, ferritin-like material and FeIII oxyhydroxide nanoparticles were detected, via Mössbauer, in intact Jurkat cells and their isolated mitochondria. EPR spectroscopy was used to quantify Fe-containing species in the respiratory complexes. Contributions from heme a, b and c centers were quantified using electronic absorption spectroscopy. Results were collectively assessed to estimate the first “ironome” profile of a human cell. The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and Fe^(III) citrate (FC), and of isolated mitochondria therefrom, was characterized. On average, only 400 ± 100 Fe’s loaded per ferritin complex, regardless of the medium Fe concentration. The extent of nanoparticle formation scaled nonlinearly with the concentration of FC in the medium. Nanoparticle formation was not strongly correlated with ROS damage. Cells could utilize nanoparticles Fe, converting them into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage, and generally contained more nanoparticles. Cells grown with TBI rather than FC contained lower Fe concentrations, more ferritin and fewer nanoparticles. Frataxin-deficient cells contained more nanoparticles than comparable WT cells. Data were analyzed by a chemically-based mathematical model. Fermenting Saccharomyces cerevisiae cells grown with varying [Fe] were also studied. The high-affinity Fe import pathway was active only in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400 – 450 µM Fe. In these cells the concentration of nonmitochondrial NHHS Fe^(II) declined 3-fold, relative to in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe^(III) increased to a limiting cellular concentration of ~ 300 µM. Isolated mitochondria contained more NHHS Fe^(II) ions and substantial amounts of Fe^(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change of nutrient Fe levels. Advisors/Committee Members: Lindahl, Paul A (advisor), Barondeau, David P (committee member), Reinhart, Gregory (committee member), Polymenis, Michael (committee member).

Subjects/Keywords: iron; metabolism; trafficking; mossbauer; epr; biophysical; jurkat; mitochondria

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APA (6^th Edition):

Jhurry, N. (2013). Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/151675

Chicago Manual of Style (16^th Edition):

Jhurry, Nema. “Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae.” 2013. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/151675.

MLA Handbook (7^th Edition):

Jhurry, Nema. “Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae.” 2013. Web. 28 Feb 2021.

Vancouver:

Jhurry N. Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae. [Internet] [Doctoral dissertation]. Texas A&M University; 2013. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/151675.

Council of Science Editors:

Jhurry N. Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae. [Doctoral Dissertation]. Texas A&M University; 2013. Available from: http://hdl.handle.net/1969.1/151675

Texas A&M University

12. Chupik, Rachel Beth. Developments in the Understanding of the Dinitrosyl Iron Unit: Its Stabilization, Reactivity, and Nitric Oxide Release.

Degree: PhD, Chemistry, 2017, Texas A&M University

URL: http://hdl.handle.net/1969.1/173248

► Dinitrosyl iron complexes (DNICs), as well as S-nitrosothiols (RSNOs), form endogenously to provide a stable means of storage and transport for the highly reactive signaling… (more)

▼ Dinitrosyl iron complexes (DNICs), as well as S-nitrosothiols (RSNOs), form endogenously to provide a stable means of storage and transport for the highly reactive signaling molecule, nitric oxide. Through the development of biomimetic complexes, the fundamental chemistry of such DNICs is established within a range of ligand sets as anionic and neutral donors that stabilize the dinitrosyl iron unit, DNIU, in oxidized {Fe(NO)₂}⁹ and reduced {Fe(NO)₂}¹⁰ (Enemark-Feltham notation) redox levels. This amorphous electronic characteristic is readily accommodated by amino acid residues, cysteinyl S- and histidine N-donors, and their surrogates, as ligands to iron. My research targets a greater understanding of DNIC reactivity with components of cellular environments, the possible connections between the electronically similar d⁹ , Cu(II) and d¹⁰, Cu(I), redox couple, the DNIC/Copper/RSNO connections, structural studies of aggregates of DNICs with metallodithiolates, as well as design strategies for NO-release therapeutic development of DNICs. A stable, reduced, {Fe(NO)₂}¹⁰ DNIC containing a bipyridyl ligand, [(neo)Fe(NO)₂] (neo = 2,9-dimethyl-1,10-phenanthroline) was used to explore redox switches and N₂ ligand exchange between copper and the Fe(NO)₂. Both Cu^I and Cu^II sources were found to promote neo ligand transfer from the DNIU with concomitant NO release. With Cu^II, redox processes were also involved, as evidenced by the formation of a mixture of oxidized {Fe(NO)₂}⁹ DNICs, in addition to both Cu^I- and Cu^II-neo complexes. Copper is known to catalyze the release of NO from RSNOs, and our discoveries found here are the first biomimetic investigation of copper’s reactivity with DNICs. Reaction of an RSNO with reduced, {Fe(NO)₂}¹⁰ DNICs, [(L)₂Fe(NO)₂] (L = CO or neo), results in NO release and the formation of a unique (-S)(SR)₂[Fe(NO)₂]₄ cluster, containing an interstitial sulfide. Such a cluster can be visualized as a plausible intermediate in the known conversion of DNICs to FeS clusters. The NO released showed complete scrambling with ¹⁵N-labeled RSNO. Although DNICs are promising NO-delivery candidates, the lack of biocompatible examples has hindered their development into useful therapeutics. We reported the first DNICs with pendant thiosugars, isolated in both monomeric and dimeric form. Cytotoxicity towards endothelial cells was low and steady NO release over several hours was observed in aqueous media. Advisors/Committee Members: Darensbourg, Marcetta Y (advisor), Lindahl, Paul (committee member), Nippe, Michael (committee member), Villalobos, Alice Y. (committee member).

Subjects/Keywords: Dinitrosyl iron unit; dinitrosyl iron complex; DNIC; nitric oxide

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APA (6^th Edition):

Chupik, R. B. (2017). Developments in the Understanding of the Dinitrosyl Iron Unit: Its Stabilization, Reactivity, and Nitric Oxide Release. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/173248

Chicago Manual of Style (16^th Edition):

Chupik, Rachel Beth. “Developments in the Understanding of the Dinitrosyl Iron Unit: Its Stabilization, Reactivity, and Nitric Oxide Release.” 2017. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/173248.

MLA Handbook (7^th Edition):

Chupik, Rachel Beth. “Developments in the Understanding of the Dinitrosyl Iron Unit: Its Stabilization, Reactivity, and Nitric Oxide Release.” 2017. Web. 28 Feb 2021.

Vancouver:

Chupik RB. Developments in the Understanding of the Dinitrosyl Iron Unit: Its Stabilization, Reactivity, and Nitric Oxide Release. [Internet] [Doctoral dissertation]. Texas A&M University; 2017. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/173248.

Council of Science Editors:

Chupik RB. Developments in the Understanding of the Dinitrosyl Iron Unit: Its Stabilization, Reactivity, and Nitric Oxide Release. [Doctoral Dissertation]. Texas A&M University; 2017. Available from: http://hdl.handle.net/1969.1/173248

Texas A&M University

13. Martin Jr., Darrell Wayne. A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases.

Degree: PhD, Chemistry, 2016, Texas A&M University

URL: http://hdl.handle.net/1969.1/187371

► Hydrogen has received widespread attention as a potential energy carrier due to its high energy content and clean combustion product H2O. [FeFe]-H2ases exhibit the highest… (more)

▼ Hydrogen has received widespread attention as a potential energy carrier due to its high energy content and clean combustion product H2O. [FeFe]-H2ases exhibit the highest H2 production rates utilizing a complex iron sulfur cofactor, called the H-cluster that requires three biosynthetic maturation proteins. Mutagenesis studies of conserved residues surrounding the H-cluster led to variants with either decreased overall activity or minimal H-cluster incorporation. These studies underscore the importance of the protein matrix in tuning active site chemistry. Here, we investigated a new class of enzymes consisting of N-terminal [FeFe]-H2ase fused to a C-terminal rubrerythrin domain (named HydY). Using protein film electrochemistry and colorimetric assays, HydY was found to function differently than standard [FeFe]-H2ases: it exhibits strong product inhibition for H+ reduction, a low KM for H2 oxidation, bias toward H2 oxidation and significant overpotential. We hypothesized that the altered reactivity for HydY was due to hydrogen bonds from conserved residue substitutions vital for H-cluster coordination. Consistent with this hypothesis, HydY variants result in enzymes with catalytic properties more similar to traditional [FeFe]-H2ases. In addition, the C-terminal domain (CTD) of HydY efficiently reduces H2O2 to H2O. Electronic absorbance, EPR and Mӧssbauer spectroscopic studies of the CTD are consistent with a rubrerythrin diiron active site with flanking mononuclear iron sites. A 1.77 Å crystal structure of the CTD reveals a domain swapped dimer in which ligands for a modified di-iron rubrerythrin active site are provided by residues across the dimer interface. Further, our results indicate that electrons generated by the oxidation of H2 are transferred to the CTD, presumably for H2O2 reduction. This is the first example of H2-dependent peroxidase. We hypothesize that evolution ‘tuned’ HydY to favor H2 oxidation and that HydY has a protective role in anaerobic bacteria that allows survival upon transient oxygen exposure. Additional bioinformatics indicate that HydY belongs to a broader class of [FeFe]-H2ases that also use H2 as a reductant for various substrates. Overall, these studies identify determinants for controlling active site chemistry of [FeFe]-H2ases that may lead to improved design of biomimetic compounds with implications in energy production. Advisors/Committee Members: Barondeau, David P (advisor), Darensbourg, Marcetta Y (committee member), Lindahl, Paul A (committee member), Raushel, Frank M (committee member).

Subjects/Keywords: hydrogenase; rubrerythrin; rubredoxin; protein film electrochemistry; catalytic bias; peroxidase

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APA (6^th Edition):

Martin Jr., D. W. (2016). A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/187371

Chicago Manual of Style (16^th Edition):

Martin Jr., Darrell Wayne. “A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases.” 2016. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/187371.

MLA Handbook (7^th Edition):

Martin Jr., Darrell Wayne. “A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases.” 2016. Web. 28 Feb 2021.

Vancouver:

Martin Jr. DW. A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases. [Internet] [Doctoral dissertation]. Texas A&M University; 2016. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/187371.

Council of Science Editors:

Martin Jr. DW. A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases. [Doctoral Dissertation]. Texas A&M University; 2016. Available from: http://hdl.handle.net/1969.1/187371

Texas A&M University

14. Park, Jinkyu. Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach.

Degree: PhD, Chemistry, 2013, Texas A&M University

URL: http://hdl.handle.net/1969.1/151831

► Fe metabolism in budding yeast Saccharomyces cerevisiae was studied using an integrative systems-level approach involving Mӧssbauer, EPR, UV-Vis spectroscopy and LC-ICP-MS, combined with conventional biochemical… (more)

▼ Fe metabolism in budding yeast Saccharomyces cerevisiae was studied using an integrative systems-level approach involving Mӧssbauer, EPR, UV-Vis spectroscopy and LC-ICP-MS, combined with conventional biochemical techniques. Wild-type cells growing exponentially on rich and minimal media were well-regulated in terms of cellular Fe homeostasis, while post-exponentially grown cells were unregulated. Such cells became overloaded with Fe^(III) oxyhydroxide nanoparticles and nonheme high spin (NHHS) Fe^(III). Fe overloading probably arose from a mismatch between growth rate and Fe uptake rate. A mathematical model that describes iron trafficking and regulation in these cells was developed. The speciation of Fe in cells also depended on the nutrient composition of the growth media. Adenine deficiency induced a transient reduction of vacuolar Fe^(III) to Fe^(II) which probably accumulated in the cytosol. The concentration of glucose impacted the Fe import rate but had little effect on Fe speciation. The concentration of amino acids and nucleotide bases impacted the level of Fe accumulation and shifted the Fe distribution toward NHHS Fe^(II). A thermodynamic model which correlated nutrient-dependent Fe transformations with vacuolar pH and redox status was developed. The effect of deleting the MTM1 gene, which encodes a transport carrier on the mitochondrial inner membrane, was investigated. Deleting MTM1 caused Fe to accumulate in mitochondria and the Mn superoxide dismutase 2 (SOD2) activity to decline. Previous studies had concluded that this inactivation arose from the misincorporation of Fe into apo-Sod2p. Most of the accumulated Fe was found to be Fe^(III) nanoparticles which are unlikely to misincorporate into apo-Sod2p. Soluble extracts from WT and ∆mtm1 mitochondria were subjected to size-exclusion and anion-exchange liquid chromatography interfaced with an on-line ICP-MS. Two major Mn peaks were observed, one due to MnSod2p and the other to a Mn species with a molecular mass of 2 - 3 kDa. None of the Fe traces comigrated precisely with MnSod2p, contrary to the Fe-misincorporation hypothesis. Deleting MTM1 probably diminishes SOD2 activity by failing to metallate apo-Sod2 protein. The low-molecular-mass Mn species may function to install Mn into apo-Sod2p during maturation in the mitochondrial matrix, using some maturation factor imported by Mtm1p. Advisors/Committee Members: Lindahl, Paul A (advisor), Begley, Tadhg P (committee member), Raushel, Frank M (committee member), Reinhart, Gregory D (committee member).

Subjects/Keywords: Saccharomyces cerevisiae; iron; Mӧssbauer; growth mode; nutrient; mtm1

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APA (6^th Edition):

Park, J. (2013). Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/151831

Chicago Manual of Style (16^th Edition):

Park, Jinkyu. “Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach.” 2013. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/151831.

MLA Handbook (7^th Edition):

Park, Jinkyu. “Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach.” 2013. Web. 28 Feb 2021.

Vancouver:

Park J. Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach. [Internet] [Doctoral dissertation]. Texas A&M University; 2013. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/151831.

Council of Science Editors:

Park J. Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach. [Doctoral Dissertation]. Texas A&M University; 2013. Available from: http://hdl.handle.net/1969.1/151831

Texas A&M University

15. Bethel, Ryan D. The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes.

Degree: PhD, Chemistry, 2014, Texas A&M University

URL: http://hdl.handle.net/1969.1/153826

► The discovery of a diiron organometallic active site, found in the [FeFe]-Hydrogenase (H2ase) enzyme, has led to a revisiting of the classic organometallic chemistry involving… (more)

▼ The discovery of a diiron organometallic active site, found in the [FeFe]-Hydrogenase (H2ase) enzyme, has led to a revisiting of the classic organometallic chemistry involving the Fe-Fe bond and bridging ligands. This diiron site is connected to a mainstay of biochemistry, a redox active 4Fe4S cluster, and the combination of these units is undoubtedly connected to the enzyme’s performance. The regioselectivity of CO substitution on the diiron framework of the so-called parent model complex (μ-pdt)[Fe(CO)3]2, (pdt = propane-1,3-dithiolate), and its derivatives, informs on the interplay of electron density in the diiron core of the enzyme active site. The structural isomers (μ-pdt)[Fe(NHC)(NO)(PMe3)][Fe(CO)3]+ and (μ-pdt)(μ-CO)[Fe(NHC)(NO)][Fe(PMe3)(CO)2]+, synthesized through CO substitution by opposing nucleophilic (PMe3) and electrophilic (NO+) ligands provide insight into the reactivity of both irons as a function of their π-acidity. The intramolecular fluxional processes of a series of (μ-SRS)[Fe(CO)3]2 complexes allows for the generation of an open site mimicking the structure of the H2ase where H+ binds in the catalytic cycle of H2 production. Density Functional Theory (DFT) was used to support the dynamic 1H and 13C NMR spectroscopic studies that established the energy barriers to both the chair/boat interconversion of FeS2C2X, where X = NR or CR2, and the rotation of the Fe(CO)3 moiety, a process essential to the formation of an open site. It was determined that the rotation barrier is correlated with the steric bulk of the bridging ligand that can be directed towards the iron. This is seen with the methyl substituent in both N(CH3) and C(CH3)2 producing a lower barrier to Fe(CO)3 rotation than the NH and CH2 analogues, while the steric bulk of NC(CH3)3 cannot be directed to the iron and results in a higher barrier than both NH and N(CH)3. Another class of bioorganometallic molecules, the dinitrosyl iron complexs (DNICs), is formed in vivo as the product of NO degradation of iron-sulfur clusters; DNICs are thought to have possible NO storage and transport roles in the body. Computational investigations utilizing DFT have been used to support synthetic and kinetic studies of the reactivity of one such complex, (NHC)(SPh)Fe(NO)2, (NHC = N-heterocyclic carbene) with CO. Advisors/Committee Members: Darensbourg, Marcetta Y (advisor), Hall, Michael B (committee member), Lindahl, Paul A (committee member), Igumenova, Tatyana I (committee member).

Subjects/Keywords: Organometallic; Hydrogenase; DNIC

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APA (6^th Edition):

Bethel, R. D. (2014). The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/153826

Chicago Manual of Style (16^th Edition):

Bethel, Ryan D. “The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes.” 2014. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/153826.

MLA Handbook (7^th Edition):

Bethel, Ryan D. “The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes.” 2014. Web. 28 Feb 2021.

Vancouver:

Bethel RD. The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes. [Internet] [Doctoral dissertation]. Texas A&M University; 2014. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/153826.

Council of Science Editors:

Bethel RD. The Bioorganometallic Chemistry of Iron and the Diatomic Ligands CO and NO as Related to Hydrogenase Active Sites and Dinitrosyl Iron Complexes. [Doctoral Dissertation]. Texas A&M University; 2014. Available from: http://hdl.handle.net/1969.1/153826

Texas A&M University

16. McCormick, Sean P. The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems.

Degree: PhD, Chemistry, 2014, Texas A&M University

URL: http://hdl.handle.net/1969.1/154123

► Eukaryotic cells contain low-molecular-mass metal complexes (LMMMCs), defined as having masses between 200 – 10,000 Da, but these so-called labile or chelatable metal pools are… (more)

▼ Eukaryotic cells contain low-molecular-mass metal complexes (LMMMCs), defined as having masses between 200 – 10,000 Da, but these so-called labile or chelatable metal pools are poorly defined in terms of structures and functions. LMMMCs are thought to participate in metal-ion regulation, trafficking, storage and/or signaling in cells. These cellular processes are often dysfunctional in metal-associated diseases. The objective of these studies was to detect and characterize LMMMCs in eukaryotic cells, organelles and tissues. A novel liquid chromatography system in a cold inert-atmosphere glove box was interfaced with an in-line inductively coupled plasma mass spectrometer, and this LC-ICP-MS system was used to detect LMMMCs in yeast cells, mitochondria, and vacuoles as well as in mouse brain and liver cells and mitochondria. In each biological system, this separations technique was applied to detect numerous LMMMCs. The molecular mass and concentration of such species were estimated. In yeast, the previously reported mismetallation of MnSOD2 was examined in the mutant strain Δmtm1. A combination of SEC and AEX chromatography revealed that the degree of mismetallation of the SOD2 protein, in which Fe replace Mn in the active site, was no greater in Δmtm1 cells than in WT cells. The mitochondria of such mutant cells did exhibit an intense chromatography peak of Mn corresponding to at mass of 2000 – 3000 Da. Mitochondria from WT cells exhibited a similar species, but at much lower intensity. This was the only Mn species present, suggesting that it was the used to metallate apo-SOD2. Mitochondria isolated from WT yeast cells contained 6 Co, 3 Cu, 2 Mn, 5 Fe and 3 Zn LMMMCs and approximately 6 P- and S- LLM species. Some of the P- and S- LMMCs probably arose from compounds like ATP, ADP, etc. Molecular masses of the LMM Cu peaks were higher (> 5 kDa) than for the LMM complexes of other transition metals. Zinc, Mn, and Fe had multiple species of interest which demonstrate the presence and labiality of the metals in pools. The same separation system was utilized to examine mice brain LMM extracts were found to contain > 30 LMMMCs. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn LLM complexes were detected. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4–20 kDa. In these systems, Co was the only metal for which the aqueous complex was reproducibly observed. A second mouse study used the LC-ICP-MS system to examine the forms of iron present in mouse plasma. Chromatograms exhibited ~6 Fe-associated peaks that were assigned to ferritin, transferrin, and hemopexin, respectively; the other 3 peaks could not be assigned. The LC-ICP-MS experiment demonstrates that numerous Fe-containing species coexist with transferrin in healthy WT mouse plasma. Advisors/Committee Members: Lindahl, Paul A (advisor), Barondeau, David P (committee member), Hilty, Christian (committee member), Pellois, Jean-Philippe (committee member).

Subjects/Keywords: Labile Metal Pools; Metal Pools; LC-ICP-MS; Metal Speciation; Yeast; Mice Brain; mtm1p; MnSOD; metal trafficking; metal homeostasis; Bioanalytical; Manganese Pool; Low Molecular Mass Metal Complexes; LMM; LMMMCs

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APA (6^th Edition):

McCormick, S. P. (2014). The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/154123

Chicago Manual of Style (16^th Edition):

McCormick, Sean P. “The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems.” 2014. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/154123.

MLA Handbook (7^th Edition):

McCormick, Sean P. “The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems.” 2014. Web. 28 Feb 2021.

Vancouver:

McCormick SP. The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems. [Internet] [Doctoral dissertation]. Texas A&M University; 2014. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/154123.

Council of Science Editors:

McCormick SP. The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems. [Doctoral Dissertation]. Texas A&M University; 2014. Available from: http://hdl.handle.net/1969.1/154123

Texas A&M University

17. Huang, Ying. Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation.

Degree: PhD, Chemistry, 2012, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10730

► With the objective to functionalize proteins for the understanding of their biological roles and developing protein-based biosensors, I have been developing methods to synthesize proteins… (more)

▼ With the objective to functionalize proteins for the understanding of their biological roles and developing protein-based biosensors, I have been developing methods to synthesize proteins with defined modifications and applying them to study protein functional roles and generate proteins with new properties. These methods rely on the read-through of an in-frame stop codon in mRNA by a nonsense suppressor tRNA specifically acylated with a noncanoncial amino acid (NAA) by a unique aminoacyl-tRNA synthetase and the genetic incorporation of this NAA at the stop codon site. NAAs either provide chemical handles for site-specific manipulation or mimic the posttranslational modifications, which are critical for understanding cellular regulations and signal transduction. The pyrrolysine synthetase (PylRS) has been wildly used to incorporate NAAs into proteins in E. coli. Taking advantage of PylRS, I have developed method to genetically incorporate ketone-containing N--acetyl-L-lysine analog, 2-amino-8-oxononanoic acid (KetoK), into proteins for their site-specific modifications and used it to mimic the protein lysine acetylation process. I have also modified the ribosome in order to improve the amber suppression efficiency and therefore to achieve incorporation of multiple copies of NAA into one protein. By overexpressing a truncated ribosomal protein, L11C, I have demonstrated 5-fold increase of amber suppression level in E. coli, leading to higher expression levels for proteins incorporated with NAAs. I have also demonstrated this method can be applied successfully to incorporate at least 3 NAAs into one protein in E. coli. With the success of incorporating multiple NAAs into one protein, I have further introduced two distinct NAAs into one protein simultaneously. This is done by using a wild type or evolved PylRS-pylTUUA pair and an evolved M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS)-tRNACUA pair. By suppressing both UAG and UAA stop codons in one mRNA, a protein incorporated with two NAAs is synthesized with a decent yield. There is of great interest to incorporate new NAAs into proteins, which is done by library selection. By introducing both positive and negative selective markers into one plasmid, I have developed a one-plasmid selection method. In this method, the positive and negative selections are accomplished by in a single type of cells hosting a single selection plasmid. Advisors/Committee Members: Liu, Wenshe (advisor), Lindahl, Paul A. (committee member), Watanabe, Coran M. (committee member), Yang, Jiong (committee member).

Subjects/Keywords: Noncanonical Amino Acids; PylRS

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APA (6^th Edition):

Huang, Y. (2012). Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10730

Chicago Manual of Style (16^th Edition):

Huang, Ying. “Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation.” 2012. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10730.

MLA Handbook (7^th Edition):

Huang, Ying. “Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation.” 2012. Web. 28 Feb 2021.

Vancouver:

Huang Y. Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation. [Internet] [Doctoral dissertation]. Texas A&M University; 2012. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10730.

Council of Science Editors:

Huang Y. Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation. [Doctoral Dissertation]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-10730

Texas A&M University

18. Zeng, Haifeng. Quantitative Determination of Chemical Processes by Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance Spectroscopy.

Degree: PhD, Chemistry, 2012, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-11103

► Dissolution dynamic nuclear polarization (DNP) provides several orders of magnitude of NMR signal enhancement by converting the much larger electron spin polarization to nuclear spin… (more)

▼ Dissolution dynamic nuclear polarization (DNP) provides several orders of magnitude of NMR signal enhancement by converting the much larger electron spin polarization to nuclear spin polarization. Polarization occurs at low temperature (1.4K) and is followed by quickly dissolving the sample for room temperature NMR detection. DNP is generally applicable to almost any small molecules and can polarize various nuclei including 1H, 19F and 13C. The large signal from DNP enhancement reduces the limit of detection to micromolar or sub-micromolar concentration in a single scan. Since DNP enhancement often provides the only source for the observable signal, it enables tracking of the polarization flow. Therefore, DNP is ideal for studying chemical processes. Here, quantitative tools are developed to separate kinetics and spin relaxation, as well as to obtain structural information from these measurements. Techniques needed for analyzing DNP polarized sample are different from those used in conventional NMR because a large, yet non-renewable hyperpolarization is available. Using small flip angle pulse excitation, the hyperpolarization can still be divided into multiple scans. Based on this principle, a scheme is presented that allows reconstruction of indirect spectral dimensions similarly to conventional 2D NMR. Additionally, small flip angle pulses can be used to obtain a succession of scans separated in time. A model describing the combined effects of the evolution of a chemical process and of spin-lattice relaxation is shown. Applied to a Diels-Alder reaction, it permitted measuring kinetics along with the effects of auto- and cross-relaxation. DNP polarization of small molecules also shows significant promise for studying protein-ligand interaction. The binding of fluorinated ligands to the protease trypsin was studied through the observation of various NMR parameter changes, such as line width, signal intensity and chemical shift of the ligands. Intermolecular polarization transfer from hyperpolarized ligand to protein can further provide information about the binding pocket of the protein. As an alternative to direct observation of protein signal, a model is presented to describe a two-step intermolecular polarization transfer between competitively binding ligands mediated through the common binding pocket of the protein. The solutions of this model relate the evolution of signal intensities to the intermolecular cross relaxation rates, which depend on individual distances in the binding epitope. In summary, DNP provides incomparable sensitivity, speed and selectivity to NMR. Quantitative models such as those discussed here enable taking full advantage of these benefits for the study of chemical processes. Advisors/Committee Members: Hilty, Christian (advisor), Lindahl, Paul A. (committee member), Lucchese, Robert R. (committee member), Ji, Jim(Xiuquan) (committee member).

Subjects/Keywords: DNP; NMR; Kinetics; Spin Relaxation; Protein Ligand Interaction

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APA (6^th Edition):

Zeng, H. (2012). Quantitative Determination of Chemical Processes by Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance Spectroscopy. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-11103

Chicago Manual of Style (16^th Edition):

Zeng, Haifeng. “Quantitative Determination of Chemical Processes by Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance Spectroscopy.” 2012. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-11103.

MLA Handbook (7^th Edition):

Zeng, Haifeng. “Quantitative Determination of Chemical Processes by Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance Spectroscopy.” 2012. Web. 28 Feb 2021.

Vancouver:

Zeng H. Quantitative Determination of Chemical Processes by Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance Spectroscopy. [Internet] [Doctoral dissertation]. Texas A&M University; 2012. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-11103.

Council of Science Editors:

Zeng H. Quantitative Determination of Chemical Processes by Dynamic Nuclear Polarization Enhanced Nuclear Magnetic Resonance Spectroscopy. [Doctoral Dissertation]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-05-11103

Texas A&M University

19. Kamat, Siddhesh. Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway.

Degree: PhD, Chemistry, 2012, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11551

► Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine. Mechanistic characterization of ADE from Escherichia coli was performed along with biophysical studies. The structure… (more)

▼ Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine. Mechanistic characterization of ADE from Escherichia coli was performed along with biophysical studies. The structure of ADE was solved from A. tumefaciens. The structure, along with the biochemical and biophysical characterization, enabled the elucidation of the mechanism of the deaminase reaction of ADE. Elucidation of the origin of the oxygenation reactions within ADE led to the discovery of a promiscuous catalase reaction. The diiron ADE from all tested bacterial species exhibited this unusual reaction, along with the generation of superoxide and hydroxyl radicals, the latter being responsible for the oxygenation of the protein. The residues that were identified to be oxygenated were primarily the metal binding residues implying the origin of this reaction was the binuclear iron center. A group of bacterial enzymes that are co-localized in the same genomic operon as ADE but of unknown function were identified. The enzyme Bh0637 from Bacillus halodurans, a representative member of this group of enzymes was characterized. This enzyme was shown to preferentially catalyze the deamination of epigenetic base, N-6-methyadenine. Lastly, gram-negative bacteria have a highly conserved phn operon composed of 14 genes to break the C-P bond of inert alkylphosphonates. The genes phnGHIJKLM are absolutely critical for this activity. We discovered that methylphosphonate reacts first with MgATP to form alpha-D-ribose-1-methylphosphonate-5-triphosphate (RPnTP) and adenine by the action of PhnI, PhnG, PhnH and PhnL. PhnI by itself was shown to perform a novel nucleosidase reaction converting MgATP to ribose-5-triphosphate and adenine. The triphosphate moiety of RPnTP is then hydrolyzed to pyrophosphate and alpha-D-ribose-1-methylphosphonate-5-phosphate (PRPn) by PhnM. The carbon-phosphorus bond of PRPn is subsequently cleaved via a radical-based reaction to alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) and methane in the presence of S-adenosyl-L-methionine by PhnJ. Advisors/Committee Members: Raushel, Frank M. (advisor), Lindahl, Paul A. (committee member), Barondeau, David P. (committee member), Pellois, Jean-Philippe (committee member).

Subjects/Keywords: Adenine Deaminase; 6-MAD; C-P lyase; Catalase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Kamat, S. (2012). Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11551

Chicago Manual of Style (16^th Edition):

Kamat, Siddhesh. “Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway.” 2012. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11551.

MLA Handbook (7^th Edition):

Kamat, Siddhesh. “Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway.” 2012. Web. 28 Feb 2021.

Vancouver:

Kamat S. Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway. [Internet] [Doctoral dissertation]. Texas A&M University; 2012. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11551.

Council of Science Editors:

Kamat S. Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway. [Doctoral Dissertation]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11551

Texas A&M University

20. Holmes-Hampton, Gregory. Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains.

Degree: PhD, Chemistry, 2012, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11418

► Iron is essential in nearly all organisms. It is a cofactor in many proteins and enzymes. This transition metal can also be toxic because it… (more)

▼ Iron is essential in nearly all organisms. It is a cofactor in many proteins and enzymes. This transition metal can also be toxic because it participates in reactions which produce reactive oxygen species. To avoid these toxic effects while still being used for essential processes, the cell must regulate tightly iron import, metabolism, trafficking, and homeostasis. These processes were studied using biophysical methods centered on Mossbauer spectroscopy supplemented by electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. This integrated biophysical approach was applied to yeast cells, isolated yeast mitochondria, and mouse brains. We determined the concentration of Fe, and the proportion of that Fe present as iron-sulfur clusters, heme centers, mononuclear nonheme centers, and as Fe3+ oxyhydroxide (phosphate) nanoparticles for each system. In yeast, the dependence of metabolic mode of growth and iron in the growth medium on this distribution was studied. Approximately three-quarters of the iron in fermenting cells was located in vacuoles, where it was present as high-spin mononuclear Fe3+ species with rhombic symmetry. The remaining quarter was present in the mitochondria. In fermenting mitochondria 4 distinct species of iron were observed, including [Fe4S4]2+ clusters and low-spin Fe2+ hemes arising from respiratory complexes, non-heme high spin (NHHS) Fe2+ species, high spin nonheme Fe3+ species, and nanoparticles. These distributions (in both the cells and mitochondria) change when the cells are grown on iron deficient medium but remained relatively unaltered as iron in the growth medium was increased. Respiring cells had less Fe associated with vacuoles, and more Fe present as HS Fe2+. Respiring mitochondria contain more [Fe4S4]2+ clusters and low-spin Fe2+ hemes, more S = 1/2 [Fe2S2]1+ clusters, and less NHHS Fe2+, HS Fe3+ species and Fe3+ nanoparticles. These changes were rationalized by assuming that the NHHS Fe2+ and Fe3+ species, and the nanoparticles were in equilibrium within the matrix of the mitochondria, and that the Fe2+ species served as feedstock for the synthesis of iron-sulfur clusters and heme centers. The iron in the mouse brain consisted mostly of [Fe4S4]2+ clusters and Fe2+ hemes from mitochondria respiratory complexes, and of ferritin, an Fe storage protein complex. NHHS Fe2+ and Fe3+ species were also observed. The ratio of stored Fe to mitochondrial Fe was sensitive to age. The brains of prenatal animals were dominated by ferritin. Following birth up to the first 4 weeks of life, there was an increase in mitochondrial Fe and a decline of ferritin Fe. Beyond 4 weeks up to 58 weeks, levels of ferritin increased and mitochondrial Fe remained constant. The brains of mice fed an Fe-deficient diet were also studied; most of the Fe in these brains was present as mitochondrial Fe, with little stored as ferritin. A model was developed to explain these changes. Advisors/Committee Members: Lindahl, Paul A. (advisor), Cremer, Paul S. (committee member), Raushel, Frank M. (committee member), Hilty, Christian B. (committee member).

Subjects/Keywords: Iron-ome; Iron Trafficking; Iron Metabolism; Iron Homeostasis; Mössbauer spectroscopy

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APA (6^th Edition):

Holmes-Hampton, G. (2012). Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11418

Chicago Manual of Style (16^th Edition):

Holmes-Hampton, Gregory. “Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains.” 2012. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11418.

MLA Handbook (7^th Edition):

Holmes-Hampton, Gregory. “Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains.” 2012. Web. 28 Feb 2021.

Vancouver:

Holmes-Hampton G. Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains. [Internet] [Doctoral dissertation]. Texas A&M University; 2012. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11418.

Council of Science Editors:

Holmes-Hampton G. Biophysical Probes of Iron Metabolism in Yeast Cells, Mitochondria, and Mouse Brains. [Doctoral Dissertation]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2012-08-11418

Texas A&M University

21. Nguyen, Tinh T. Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily.

Degree: PhD, Chemistry, 2011, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7237

► The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A… (more)

▼ The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A more divergent member of this superfamily, URI (uronate isomerase) from Escherichia coli, catalyzes the isomerization of D-glucuronate to D-fructuronate and D-galacturonate to D-tagaturonate. In Bacillus halodurans, two distinct operons were identified for the metabolism of D-glucuronate and D-galacturonate based on kinetics and genomic context. The canonical uronate isomerase is encoded by the gene Bh0705. A second URI in this organism, Bh0493, is the outlier of the group in terms of sequence similarity. Kinetic evidences indicate that Bh0705 is relatively specific for the isomerization of D-glucuronate, while Bh0493 is specific for the Dgalacturonate pathway. Bell-shaped pH-rate profiles were observed for the wild type URI from Escherichia coli. Primary isotope effects with [2-2H]-D-glucuronate and solvent viscosity studies are consistent with product release as the rate limiting step. X-ray structure of Bh0493 was determined in the presence of D-glucuronate. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-357. Another divergent member of the AHS is the enzyme renal dipeptidase. Structural studies of the enzyme from Streptomyces coelicolor (Sco3058) demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for both Zn2+ and Cd2+ enzymes. A chemical mechanism for renal dipeptidase is proposed based on structural analysis of the enzyme-inhibitor complex. The reaction is initiated by the polarization of the amide bond by the B-metal. Asp-320 activates the bridging hydroxide for nucleophilic attack at the peptide carbon center, forming a tetrahedral intermediate that is stabilized by the metal center and His-150. The protonated Asp-320 donates the proton to the a-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond. Advisors/Committee Members: Raushel, Frank M. (advisor), Lindahl, Paul A. (committee member), Reinhart, Gregory D. (committee member), Watanabe, Coran H. (committee member).

Subjects/Keywords: Amidohydrolase superfamily; uronate isomerase; renal dipeptidase

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APA (6^th Edition):

Nguyen, T. T. (2011). Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7237

Chicago Manual of Style (16^th Edition):

Nguyen, Tinh T. “Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily.” 2011. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7237.

MLA Handbook (7^th Edition):

Nguyen, Tinh T. “Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily.” 2011. Web. 28 Feb 2021.

Vancouver:

Nguyen TT. Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily. [Internet] [Doctoral dissertation]. Texas A&M University; 2011. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7237.

Council of Science Editors:

Nguyen TT. Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily. [Doctoral Dissertation]. Texas A&M University; 2011. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7237

Texas A&M University

22. Hall, Richard Stuart. Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes.

Degree: PhD, Chemistry, 2011, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7250

► The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and… (more)

▼ The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN. Advisors/Committee Members: Raushel, Frank M. (advisor), Lindahl, Paul A. (committee member), Barondeau, David P. (committee member), Reinhart, Gregory D. (committee member).

Subjects/Keywords: Amidohydrolase superfamily; enzyme mechanism and inhibition; evolution; function discovery; NagA; cytosine deaminase; guanine deaminase; 8-oxoguanine deaminase; isoxanthopterin deaminase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hall, R. S. (2011). Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7250

Chicago Manual of Style (16^th Edition):

Hall, Richard Stuart. “Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes.” 2011. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7250.

MLA Handbook (7^th Edition):

Hall, Richard Stuart. “Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes.” 2011. Web. 28 Feb 2021.

Vancouver:

Hall RS. Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes. [Internet] [Doctoral dissertation]. Texas A&M University; 2011. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7250.

Council of Science Editors:

Hall RS. Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes. [Doctoral Dissertation]. Texas A&M University; 2011. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7250

Texas A&M University

23. Tsai, Ping-Chuan. Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents.

Degree: PhD, Chemistry, 2011, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7428

► The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta possess very broad substrate specificity for organophosphorus compounds. It is capable of hydrolyzing several insecticides including paraoxon and… (more)

▼ The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta possess very broad substrate specificity for organophosphorus compounds. It is capable of hydrolyzing several insecticides including paraoxon and various chemical warfare agents such as sarin (GB), soman (GD), cyclosarin (GF) and VX. The catalytic ability of PTE for the hydrolysis of paraoxon is close to the limit of diffusion of the reactant in solution. However, the catalytic activity of PTE for the organophosphate nerve agents is lower than that for paraoxon. It was reported that the wild-type PTE preferentially catalyze the hydrolysis of the less toxic Rp- enantiomers of organophosphate nerve agents and their analogues than the more toxic Sp- enantiomers. The first generation of PTE mutants that contains a modified substrate binding pocket was identified and it was observed that their catalytic activity towards the more toxic Sp- enantiomers organophosphate nerve agent analogues was enhanced. The H254G/H257W/L303T mutant was shown to have a reversed stereoselectivity. The kcat/Km values of this mutant towards the hydrolysis of the SpRc- and SpSc-enantiomers of the GD analogue and the Sp-enantiomer of the GF analogue were enhanced by 73-, 543-, and 1340-fold relatively to the wile-type enzyme, respectively. The second generation of PTE mutants were isolated and shown to have higher activity toward the Sp-enantiomers of the GD and GF analogues than the first generation mutants. Saturation mutagenesis, in vitro screening and in vivo selection were conducted using the gene for the mutants from the first generation. The GWT-d3 mutant was identified as the most active PTE mutant towards the hydrolysis of the Spenantiomers of the GD analogue, the kcat/Km values were 780- and 3530-fold higher than the wild-type enzyme toward the SpRc- and SpSc-enantiomers of the GD analogues. The GWT-f5 mutant was the best PTE mutant towards the Sp-enantiomer of the GF analogue, the kcat/Km values were 15500-fold higher than the wild-type enzyme. The X-ray crystal structures of the wild-type PTE and the G60A mutant were determined in the presence of the hydrolysis product diethyl phosphate and a product analogue cacodylate, respectively. This result supports the reaction mechanism previously proposed by Dr. Sarah Aubert. Advisors/Committee Members: Raushel, Frank M. (committee member), Lindahl, Paul A. (committee member), Darensbourg, Marcetta Y. (committee member), Reinhart, Gregory D. (committee member).

Subjects/Keywords: Phosphotriesterase (PTE)

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APA (6^th Edition):

Tsai, P. (2011). Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7428

Chicago Manual of Style (16^th Edition):

Tsai, Ping-Chuan. “Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents.” 2011. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7428.

MLA Handbook (7^th Edition):

Tsai, Ping-Chuan. “Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents.” 2011. Web. 28 Feb 2021.

Vancouver:

Tsai P. Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents. [Internet] [Doctoral dissertation]. Texas A&M University; 2011. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7428.

Council of Science Editors:

Tsai P. Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents. [Doctoral Dissertation]. Texas A&M University; 2011. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7428

Texas A&M University

24. Browne, Daniel R. Systems Analysis of Metabolism and Physiology of the Oil-Producing Green Alga Botryococcus braunii Race B (Showa).

Degree: PhD, Biochemistry, 2018, Texas A&M University

URL: http://hdl.handle.net/1969.1/174550

► The colony-forming green microalga Botryococcus braunii is mostly known for its ability to produce an abundance of liquid hydrocarbons. However, geochemical studies have found fossilized… (more)

▼ The colony-forming green microalga Botryococcus braunii is mostly known for its ability to produce an abundance of liquid hydrocarbons. However, geochemical studies have found fossilized remains of the species in petroleum source rocks from around the planet, dating as far back as the Precambrian eon. Thus B. braunii is considered a source of petroleum throughout the geological ages and presents an interesting model to study hydrocarbon metabolism. To better understand the biochemical and genetic systems that underpin the unique properties of B. braunii, we have sequenced and analyzed its genome. Using a comparative genomics approach, we identified 187 functions that are unique in B. braunii among the Viridiplantae (green algae and land plants), and 402 functions that are unique in B. braunii among the green algae. Thus B. braunii shares 215 functions with land plants that other green algae do not. These functions include parts of the photosynthetic apparatus, the ubiquitin system, cytochrome P450s, peptidases, cytoskeleton proteins, and others. To further understand the active interpretation of genomic information, we sequenced the transcriptome of B. braunii every six hours over the course of three days. The goal of this experiment was to determine the gene expression patterns associated with light/dark transitions. Interestingly, we found several strong coexpression modules that cycle, not according to light or dark conditions, but by time of day, indicating the presence of circadian regulatory mechanisms. To determine the impact of gene expression and time of day on metabolism, we generated metabolomics data for each of the biological samples that were utilized to obtain the transcriptome data. Targeted and untargeted analyses of polar and nonpolar metabolites revealed that unlike transcription, metabolite pools do not appear to significantly change with time of day. The information presented in this dissertation adds great value to our fundamental understanding of the systems governing B. braunii metabolism and physiology. With this knowledge, we could design genetic systems in heterologous hosts to mimic the properties of B. braunii pathways. This could result in synthetic pathways for hydrocarbon production with strong metabolic flux, technology that is vital for the development of sustainable bioproducts. Advisors/Committee Members: Devarenne, Timothy P (advisor), Aramayo, Rodolfo A (committee member), Lindahl, Paul A (committee member), Mullet, John E (committee member).

Subjects/Keywords: Botryococcus; algae; bioinformatics; genomics; transcriptomics; metabolomics; evolution; Chlorophyta; Viridiplantae

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APA (6^th Edition):

Browne, D. R. (2018). Systems Analysis of Metabolism and Physiology of the Oil-Producing Green Alga Botryococcus braunii Race B (Showa). (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/174550

Chicago Manual of Style (16^th Edition):

Browne, Daniel R. “Systems Analysis of Metabolism and Physiology of the Oil-Producing Green Alga Botryococcus braunii Race B (Showa).” 2018. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/174550.

MLA Handbook (7^th Edition):

Browne, Daniel R. “Systems Analysis of Metabolism and Physiology of the Oil-Producing Green Alga Botryococcus braunii Race B (Showa).” 2018. Web. 28 Feb 2021.

Vancouver:

Browne DR. Systems Analysis of Metabolism and Physiology of the Oil-Producing Green Alga Botryococcus braunii Race B (Showa). [Internet] [Doctoral dissertation]. Texas A&M University; 2018. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/174550.

Council of Science Editors:

Browne DR. Systems Analysis of Metabolism and Physiology of the Oil-Producing Green Alga Botryococcus braunii Race B (Showa). [Doctoral Dissertation]. Texas A&M University; 2018. Available from: http://hdl.handle.net/1969.1/174550

25. Moore, Michael John. An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool.

Degree: PhD, Chemistry, 2017, Texas A&M University

URL: http://hdl.handle.net/1969.1/165816

► Mitochondria contain a low-molecular-mass (LMM) pool of weakly bound iron complexes, called the labile iron pool (LIP). Although its composition and biological function remain largely… (more)

▼ Mitochondria contain a low-molecular-mass (LMM) pool of weakly bound iron complexes, called the labile iron pool (LIP). Although its composition and biological function remain largely uncharacterized, the LIP has been implicated in cellular iron metabolism and disease pathogenesis. In this dissertation, results obtained from Mössbauer, EPR, and UV-Vis studies were integrated with LC-ICP-MS data to investigate the chemical nature of the mitochondrial LIP. LMM Fe, Mn, Cu, Zn, and Co complexes were detected in yeast and mammalian mitochondria. Such complexes were reproducibly observed and hypothesized to metalate mitochondrial apo-metalloproteins. The approximate mass of each complex was estimated along with its mitochondrial concentration. The predominant LMM Fe species detected in mitochondria had a mass of ~580 Da (called Fe₅₈₀) and was present in both yeast and mammalian mitochondria. Increasing the Fe concentration in the medium increased the intensity of Fe₅₈₀. Interestingly, the mitochondrial concentration of Fe₅₈₀ was ~100 µM, which was consistent with previous estimates of the mitochondrial LIP. Thus, Fe₅₈₀ was hypothesized as the cytosolic iron species that is imported into mitochondria to form the LIP. Treatment with metal chelators demonstrated that Fe₅₈₀ was labile. A yeast strain lacking mitochondrial Fe importers, Mrs3/4, was characterized using biophysical and bioanalytical methods (ΔΔ). Respiring Fe-deficient ΔΔ cells exhibited a “slow-growth” phenotype that was ameliorated under Fe-sufficient conditions. ΔΔ cells accumulated more Fe than wild-type (WT) cells, even under Fe-sufficient conditions, indicating Fe dysregulation. ΔΔ cells accumulated NHHS Feᴵᴵ and Feᴵᴵᴵ in the cytosol and vacuoles, respectively. Fe-deficient ΔΔ mitochondria accumulated Feᴵᴵᴵ oxyhydroxide nanoparticles and were devoid of central doublet ([Fe₄S₄] 2+ and LS Feᴵᴵ hemes), whereas Fe-sufficient ΔΔ mitochondria contained comparable levels of central doublet but less NHHS Feᴵᴵ relative to WT. This suggested the mitochondrial NHHS Feᴵᴵ pool was reduced in Fe-sufficient ΔΔ cells. Fe₅₈₀ was absent in Fe-deficient ΔΔ mitochondria but present under Fe-sufficient conditions. Two candidate masses {664.46 and 665.46 amu} and {685.39 and 686.39 amu} were determined for Fe₅₈₀. Viewed comprehensively, this study provides strong evidence that the mitochondrial NHHS Feᴵᴵ pool is composed predominantly of Fe₅₈₀ and that the size of this pool controls cellular Fe homeostasis. Advisors/Committee Members: Lindahl, Paul A (advisor), Barondeau, David (committee member), Raushel, Frank (committee member), Gohil, Vishal (committee member).

Subjects/Keywords: Mitochondria; Iron; Pool; Labile; Copper; Transition; Metal

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APA (6^th Edition):

Moore, M. J. (2017). An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/165816

Chicago Manual of Style (16^th Edition):

Moore, Michael John. “An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool.” 2017. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/165816.

MLA Handbook (7^th Edition):

Moore, Michael John. “An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool.” 2017. Web. 28 Feb 2021.

Vancouver:

Moore MJ. An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool. [Internet] [Doctoral dissertation]. Texas A&M University; 2017. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/165816.

Council of Science Editors:

Moore MJ. An Integrative Biophysical and Bioanalytical Approach for Investigating the Mitochondrial Labile Iron Pool. [Doctoral Dissertation]. Texas A&M University; 2017. Available from: http://hdl.handle.net/1969.1/165816

26. Garber Morales, Jessica H. Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae.

Degree: PhD, Chemistry, 2011, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7846

► An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a… (more)

▼ An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria – was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups. Advisors/Committee Members: Lindahl, Paul A. (advisor), Russell, David (committee member), Hu, James (committee member), Vingh, Gyula (committee member).

Subjects/Keywords: Mitochondria; Iron; Trafficking; cytochrome; metabolism; Yeast

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APA (6^th Edition):

Garber Morales, J. H. (2011). Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7846

Chicago Manual of Style (16^th Edition):

Garber Morales, Jessica H. “Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae.” 2011. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7846.

MLA Handbook (7^th Edition):

Garber Morales, Jessica H. “Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae.” 2011. Web. 28 Feb 2021.

Vancouver:

Garber Morales JH. Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae. [Internet] [Doctoral dissertation]. Texas A&M University; 2011. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7846.

Council of Science Editors:

Garber Morales JH. Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae. [Doctoral Dissertation]. Texas A&M University; 2011. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2010-05-7846

27. Knippa, Kevin Christopher. Role of RPB9 in RNA Polymerase II Fidelity.

Degree: PhD, Biochemistry, 2013, Texas A&M University

URL: http://hdl.handle.net/1969.1/151243

► RNA polymerase II, the polymerase responsible for transcribing protein coding genes in eukaryotes, possesses an ability to discriminate between correct (complementary to the DNA template)… (more)

▼ RNA polymerase II, the polymerase responsible for transcribing protein coding genes in eukaryotes, possesses an ability to discriminate between correct (complementary to the DNA template) and incorrect substrates (selectivity), and as well as remove incorrect substrates that have been erroneously incorporated into the nascent RNA transcript (proofreading). Although these features of pol II are not as robust as those observed for DNA polymerases, the accurate utilization of genetic information is of obvious importance to the cell. The role of the small RNA polymerase II subunit Rpb9 in transcriptional proofreading was assessed in vitro. Transcription elongation complexes in which the 3'-end of the RNA is not complementary to the DNA template have a dramatically reduced rate of elongation, which provides a fidelity checkpoint at which the error can be removed. The efficiency of such proofreading depends on competing rates of error propagation (extending the RNA chain without removing the error) and error excision, a process that is facilitated by TFIIS. In the absence of Rpb9, the rate of error propagation is increased by 2- to 3-fold in numerous sequence contexts, compromising the efficiency of proofreading. In addition, the rate and extent of TFIIS-mediated error excision is also significantly compromised in the absence of Rpb9. In at least some sequence contexts, Rpb9 appears to enhance TFIIS-mediated error excision by facilitating efficient formation of a conformation necessary for RNA cleavage. If a transcription error is propagated by addition of a nucleotide to the mismatched 3'-end, the rate of further elongation increases but remains much slower than that of a complex with a fully base-paired RNA, which provides a second potential fidelity checkpoint. The absence of Rpb9 also affects both error propagation and TFIIS-mediated error excision at this potential fidelity checkpoint in a manner that compromises transcriptional fidelity. The trigger loop, a mobile structural element of the largest subunit of RNA polymerase II is important for maintaining fidelity. The pol II specific toxin α-amanitin targets the trigger loop, and was used to distinguish trigger loop -independent and -dependent Rpb9 functions, in vitro. Rpb9 decreases the correct nt extension rate when trigger loop movement is restricted by α-amanitin. This occurs in the context of a RNA with a matched or mismatched 3’-end, which indicates that Rpb9’s contribution to correct nt extension occurs in a manner independent of the trigger loop. In addition, the effect on mismatch extension indicates that the trigger loop is not required for Rpb9 to facilitate recognition of proofreading ‘checkpoints’ after mismatches occur. Rpb9 also decreases the rate of misincorporation, but this effect is dependent on the trigger loop. Rpb9’s role in selectivity was tested by utilizing several assays to estimate nt discrimination. Rpb9 does not have a significant effect on nt discrimination for the sequence contexts tested, which suggests the role Rpb9 plays in fidelity… Advisors/Committee Members: Peterson, David Oscar (advisor), Bryk, Mary (committee member), Lindahl, Paul (committee member), Wilson, Van (committee member).

Subjects/Keywords: Rpb9; RNA polymerase II; Fidelity; TFIIS

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APA (6^th Edition):

Knippa, K. C. (2013). Role of RPB9 in RNA Polymerase II Fidelity. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/151243

Chicago Manual of Style (16^th Edition):

Knippa, Kevin Christopher. “Role of RPB9 in RNA Polymerase II Fidelity.” 2013. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/151243.

MLA Handbook (7^th Edition):

Knippa, Kevin Christopher. “Role of RPB9 in RNA Polymerase II Fidelity.” 2013. Web. 28 Feb 2021.

Vancouver:

Knippa KC. Role of RPB9 in RNA Polymerase II Fidelity. [Internet] [Doctoral dissertation]. Texas A&M University; 2013. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/151243.

Council of Science Editors:

Knippa KC. Role of RPB9 in RNA Polymerase II Fidelity. [Doctoral Dissertation]. Texas A&M University; 2013. Available from: http://hdl.handle.net/1969.1/151243

28. Porter, Tamiko Neal. The structure and mechanism of bacterial dihydroorotase.

Degree: PhD, Chemistry, 2006, Texas A&M University

URL: http://hdl.handle.net/1969.1/3152

► Dihydroorotase (DHO) is a zinc metallo-enzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate… (more)

▼ Dihydroorotase (DHO) is a zinc metallo-enzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. The X-ray crystal structure of the enzyme was obtained at a resolution of 1.7 Ã. The pH-rate profiles for the hydrolysis of dihydroorotate or thio-dihydroorotate demonstrated that a single group of DHO must be unprotonated for maximal catalytic activity. The pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or CADmium by reconstitution of the apo-enzyme with divalent cations. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of Asp-250 resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during dihydroorotate hydrolysis. The X-ray crystal structure of DHO showed that the side-chain carboxylate of dihydroorotate is electrostatically interacting with Arg20, Asn-44 and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thioanalog of dihydroorotate, (TDO) was found to be a substrate of DHO. A comprehensive chemical mechanism for DHO was proposed based on the experimental data presented in this dissertation. Armed with this understanding of the structure-function relationship of DHO, a rational approach was used to alter the substrate specificity of the enzyme. The R20/N44/H254 mutant of DHO was obtained and found to have increased activity on dihydrouracil compared to the wild-type enzyme. The sequence of the gene PA5541 from Pseudomonas aeruginosa has a glutamine at a position where most active DHO proteins have a histidine residue. Results from the characterization of PA5541 indicate that it is a functional DHO. Advisors/Committee Members: Raushel, Frank (advisor), Reinhart, Gregory (committee member), Lindahl, Paul (committee member), DeRose, Victoria (committee member).

Subjects/Keywords: dihydroorotase

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APA (6^th Edition):

Porter, T. N. (2006). The structure and mechanism of bacterial dihydroorotase. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/3152

Chicago Manual of Style (16^th Edition):

Porter, Tamiko Neal. “The structure and mechanism of bacterial dihydroorotase.” 2006. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/3152.

MLA Handbook (7^th Edition):

Porter, Tamiko Neal. “The structure and mechanism of bacterial dihydroorotase.” 2006. Web. 28 Feb 2021.

Vancouver:

Porter TN. The structure and mechanism of bacterial dihydroorotase. [Internet] [Doctoral dissertation]. Texas A&M University; 2006. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/3152.

Council of Science Editors:

Porter TN. The structure and mechanism of bacterial dihydroorotase. [Doctoral Dissertation]. Texas A&M University; 2006. Available from: http://hdl.handle.net/1969.1/3152

29. Cummings, Jennifer Ann. D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity.

Degree: PhD, Chemistry, 2012, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-9021

► Approximately one third of the genes for the completely sequenced bacterial genomes have an unknown, uncertain, or incorrect functional annotation. Approximately 11,000 putative proteins identified… (more)

▼ Approximately one third of the genes for the completely sequenced bacterial genomes have an unknown, uncertain, or incorrect functional annotation. Approximately 11,000 putative proteins identified from the fully-sequenced microbial genomes are members of the catalytically diverse Amidohydrolase Superfamily. Members of the Amidohydrolase Superfamily separate into 24 Clusters of Orthologous Groups (cogs). Cog3653 includes proteins annotated as N-acyl-D-amino acid deacetylases (DAAs), and proteins within cog2355 are homologues to the human renal dipeptidase. The substrate profiles of three DAAs (Bb3285, Gox1177 and Sco4986) and six microbial dipeptidase (Sco3058, Gox2272, Cc2746, LmoDP, Rsp0802 and Bh2271) were examined with N-acyl-L-, N-acyl-D-, L-Xaa-L-Xaa, L-Xaa-D-Xaa and D-Xaa-L-Xaa substrate libraries. The rates of hydrolysis of the library components were determined by separating the amino acids by HPLC and quantitating the products. Gox1177 and Sco4986 hydrolyzed several N-acyl-D-amino acids, especially those where the amino acid was a hydrophobic residue. Gox1177 hydrolyzed L-Xaa-D-Xaa and N-acetyl-D-amino acids with similar catalytic efficiencies (~10⁴ M⁻¹s⁻¹). The best substrates identified for Gox1177 and Sco4986 were N-acetyl-D-Trp and N-acetyl-D-Phe, respectively. Conversely, Bb3285 hydrolyzed N-acyl-D-Glu substrates (kcat/Km ⁹́⁸ 5 x 10⁶M⁻¹s⁻¹) and, to a lesser extent, L-Xaa-D-Glu dipeptides. The structure of a DAA from A. faecalis did not help explain the substrate specificity of Bb3285. N-methylphosphonate derivatives of D-amino acids were inhibitors of the DAAs examined. The structure of Bb3285 was solved in complex with the N-methylphosphonate derivative of D-Glu or acetate/formate. The specificity of Bb3285 was due to an arginine located on a loop which varied in conformation from the A. faecalis enzyme. In a similar manner, six microbial renal dipeptidase-like proteins were screened with 55 dipeptide libraries. These enzymes hydrolyzed many dipeptides but favored L-D dipeptides. Respectable substrates were identified for proteins Bh2271 (L-Leu-D-Ala, kcat/Km = 7.4 x 10⁴ M⁻¹s⁻¹), Sco3058 (L-Arg-D-Asp, kcat/Km = 7.6 x 10⁵ M⁻¹s⁻¹), Gox2272 (L-Asn-D-Glu, kcat/Km = 4.7 x 10⁵ M⁻¹s⁻¹), Cc2746 (L-Met-D-Leu, kcat/Km = 4.6 x 10⁵ M⁻¹s⁻¹), LmoDP (L-Leu-D-Ala, kcat/Km = 1.1 x 10⁵ M⁻¹s⁻¹), Rsp0802 (L-Met-D-Leu, kcat/Km = 1.1 x 10⁵ M⁻¹s⁻¹). Phosphinate mimics of dipeptides were inhibitors of the dipeptidases. The structures of Sco3058, LmoDP and Rsp0802 were solved in complex with the pseudodipeptide mimics of L-Ala-D-Asp, L-Leu-D-Ala and L-Ala-D-Ala, respectively. The structures were used to assist in the identification of the structural determinants of substrate specificity. Advisors/Committee Members: Raushel, Frank M. (advisor), Barondeau, David P. (committee member), Lindahl, Paul A. (committee member), Reinhart, Gregory D. (committee member).

Subjects/Keywords: Jennifer Cummings; Amidohydrolase Superfamily; TIM-barrel; alpha-beta barrel; D-aminoacylase; Dipeptidase; dipeptides; enzymology; phosphinate; phosphonate; D-amino acid; Clusters of Orthologous Groups; Cc2746; Gox2272; Bb3285; Rsp_0802; Lmo2462; Bh2271; Sco4986

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Cummings, J. A. (2012). D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-9021

Chicago Manual of Style (16^th Edition):

Cummings, Jennifer Ann. “D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity.” 2012. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-9021.

MLA Handbook (7^th Edition):

Cummings, Jennifer Ann. “D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity.” 2012. Web. 28 Feb 2021.

Vancouver:

Cummings JA. D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity. [Internet] [Doctoral dissertation]. Texas A&M University; 2012. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-9021.

Council of Science Editors:

Cummings JA. D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity. [Doctoral Dissertation]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-9021

30. Miao, Ren. Probing Iron Accumulation in Sacchromyces cerevisiae Using Integrative Biophysical and Biochemical Techniques.

Degree: PhD, Chemistry, 2012, Texas A&M University

URL: http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8743

► Iron is an essential element for life. It is involved in a number of biological processes, including iron sulfur (Fe/S) cluster assembly and heme biosynthesis.… (more)

▼ Iron is an essential element for life. It is involved in a number of biological processes, including iron sulfur (Fe/S) cluster assembly and heme biosynthesis. However it is also potentially toxic due to its ability to induce formation of reactive oxygen species (ROS) via Fenton chemistry. Therefore its uptake, trafficking and utilization must be regulated to avoid its toxicological effect. It has been recently discovered that Fe/S cluster biosynthesis machinery plays a key role in the cellular iron regulation and its disruption leads to impaired iron regulation and iron accumulation within mitochondria. The iron accumulation resulted from impaired Fe/S cluster assembly in the eukaryotic model organism Saccharomyces cerevisiae (baker’s yeast) was studied. Various biophysical (e.g. Mössbauer, EPR, UV-vis spectroscopy) and biochemical (e.g. Western blots, PCR, enzyme activity assay, etc.) techniques were used to characterize the iron content in yeast mitochondria isolated from several mutants strains. In these mutants one of the proteins involved in Fe/S cluster biosynthesis (Yah1p and Atm1p) is mutated and iron regulation and metabolism are disrupted. By integrating the results obtained from these different methods, it was determined that excess iron accumulates in the mutant mitochondria as inorganic phosphate Fe(III) nano-particles exhibiting superparamagnetic behaviors. Oxygen is required for iron accumulation and nanoparticle formation. The Fe(III) nano-particles can be chemically reduced to Fe(II) then largely exported from the mitochondria. These biophysical and biochemical methods were also used to examine the iron distribution in whole yeast cells of the Aft1-1up strain in which iron regulon genes are constitutively activated and compared to that of Yah1p-depleted and wild type yeast. Constitutive activation of iron regulon genes does not alter the cellular iron distribution significantly. However disruption of Fe/S cluster assembly by Yah1p depletion causes dramatic cellular iron redistribution: the vacuolar iron is largely evacuated and most of the cellular iron probably precipitates in mitochondria as Fe(III) nanoparticles. The results provide novel insights into iron trafficking and possible signal communications between organelles within cells. Advisors/Committee Members: Lindahl, Paul A. (advisor), Barondeau, David P. (committee member), Begley, Tadhg P. (committee member), Raushel, Frank M. (committee member).

Subjects/Keywords: Biological iron; Mossbauer spectroscopy; EPR spectroscopy; Electron microscopy; XAS spectroscopy; UV-vis spectroscopy; Nanoparticles

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Miao, R. (2012). Probing Iron Accumulation in Sacchromyces cerevisiae Using Integrative Biophysical and Biochemical Techniques. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8743

Chicago Manual of Style (16^th Edition):

Miao, Ren. “Probing Iron Accumulation in Sacchromyces cerevisiae Using Integrative Biophysical and Biochemical Techniques.” 2012. Doctoral Dissertation, Texas A&M University. Accessed February 28, 2021. http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8743.

MLA Handbook (7^th Edition):

Miao, Ren. “Probing Iron Accumulation in Sacchromyces cerevisiae Using Integrative Biophysical and Biochemical Techniques.” 2012. Web. 28 Feb 2021.

Vancouver:

Miao R. Probing Iron Accumulation in Sacchromyces cerevisiae Using Integrative Biophysical and Biochemical Techniques. [Internet] [Doctoral dissertation]. Texas A&M University; 2012. [cited 2021 Feb 28]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8743.

Council of Science Editors:

Miao R. Probing Iron Accumulation in Sacchromyces cerevisiae Using Integrative Biophysical and Biochemical Techniques. [Doctoral Dissertation]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8743

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