subject:(Enzymology)

1. Petro, Elizabeth Jane. Seeking the Lipid Substrate Binding Site: Structural Studies of Eukaryotic Diacylglycerol Kinases.

Degree: 2013, Johns Hopkins University

URL: http://jhir.library.jhu.edu/handle/1774.2/37057

► Very little is known about the structure of the catalytic domain of eukaryotic diacylglycerol kinases (DGKs), a family of interfacial enzymes implicated in a number… (more)

▼ Very little is known about the structure of the catalytic domain of eukaryotic diacylglycerol kinases (DGKs), a family of interfacial enzymes implicated in a number of physiological roles and human diseases. Constructs of the catalytic domain could be expressed in Escherichia coli and extracted, refolded, and purified from inclusion bodies, but when subjected to analytical gel filtration chromatography, these constructs eluted in the void volume in what appeared to be microscopic aggregates unsuitable for x-ray crystallography. Adding glutathione S-transferase, thioredoxin, or maltose binding protein as N-terminal fusion tags did not improve the constructs’ solubility. Coexpressing with bacterial chaperones increased the yield in the supernatant after high-speed centrifugation, but the protein still eluted in the void upon analytical gel filtration chromatography. DGK constructs expressed in insect cells were likewise insoluble and unsuitable for x-ray crystallography. Loss of enzymatic activity of purified DGK could be mitigated by including 50% (v/v) glycerol and storing at -80°C, and by including detergent, lipid, and protein activators following thawing. Certain polybasic proteins and substrate analogs increase the in vitro enzymatic activity of purified DGK, whereas hirudin decreases its activity under certain conditions. DGK is able to use certain diglyceride analogs as substrates. Bimolecular fluorescence complementation was unable to show whether DGK-theta dimerized or interacted with tau when overexpressed in mammalian cells. Thrombin was able to digest DGK-theta after folding, but none of the washing conditions tested were able to separate the digested fragments, so while digestion with thrombin appeared to activate DGK-theta, whether the activation was due to relief from auto-inhibition or some other effect from the thrombin could not be resolved. Photoaffinity labels were able to enzymatically inactivate purified DGK-theta in a probe-, ultraviolet-, and time-dependent way, but only when the linker between the substrate analog moiety and the photoactivatable moiety was sufficiently short. Mass spectrometry studies were unable to detect peptides shifted by the predicted molecular weight of the photoaffinity labels or their fragments. This work should guide future studies of the structure of eukaryotic DGKs. Advisors/Committee Members: Amzel, L M (advisor).

Subjects/Keywords: Biochemistry; Enzymology

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APA (6^th Edition):

Petro, E. J. (2013). Seeking the Lipid Substrate Binding Site: Structural Studies of Eukaryotic Diacylglycerol Kinases. (Thesis). Johns Hopkins University. Retrieved from http://jhir.library.jhu.edu/handle/1774.2/37057

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Petro, Elizabeth Jane. “Seeking the Lipid Substrate Binding Site: Structural Studies of Eukaryotic Diacylglycerol Kinases.” 2013. Thesis, Johns Hopkins University. Accessed January 20, 2021. http://jhir.library.jhu.edu/handle/1774.2/37057.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Petro, Elizabeth Jane. “Seeking the Lipid Substrate Binding Site: Structural Studies of Eukaryotic Diacylglycerol Kinases.” 2013. Web. 20 Jan 2021.

Vancouver:

Petro EJ. Seeking the Lipid Substrate Binding Site: Structural Studies of Eukaryotic Diacylglycerol Kinases. [Internet] [Thesis]. Johns Hopkins University; 2013. [cited 2021 Jan 20]. Available from: http://jhir.library.jhu.edu/handle/1774.2/37057.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Petro EJ. Seeking the Lipid Substrate Binding Site: Structural Studies of Eukaryotic Diacylglycerol Kinases. [Thesis]. Johns Hopkins University; 2013. Available from: http://jhir.library.jhu.edu/handle/1774.2/37057

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Arizona

2. Nelp, Micah. Biological Synthesis and Transformation of Nitriles .

Degree: 2016, University of Arizona

URL: http://hdl.handle.net/10150/621560

► Nitrile-containing natural products are rare in Nature, and there have been very few studies on the mechanisms by which they are synthesized and utilized. The… (more)

▼ Nitrile-containing natural products are rare in Nature, and there have been very few studies on the mechanisms by which they are synthesized and utilized. The biosynthesis of 7-deazapurine containing natural products is a unique case whereby both formation of a nitrile and its conversion to an amide are documented. The overall theme of this work is to interrogate the biosynthesis of the nitrile intermediate in the pathway and its subsequent hydration to an amide. The biosynthesis 7-cyano-7-deazaguanine (preQ₀), the key intermediate in the biosynthesis of the hypermodified base queuosine and the toyocamycin natural product, is accomplished by preQ₀ synthetase through a series of unprecedented reactions whereby the carboxylate moiety of the substrate, 7-carboxy-7-deazaguanine (CDG), is successively activated by adenylation, reacted with ammonia, and dehydrated to produce the nitrile. This one-enzyme synthesis of a nitrile is unique as the only other known route to nitriles proceeds through at least two enzymes. Nitrile hydratases are metalloenzymes that selectively hydrate nitriles to the amide and are used industrially to produce acrylamide and nicotinamide. These enzymes use a trivalent iron or cobalt complex comprised of two backbone amidate ligands and three cysteine thiolate ligands of which two are modified to the sulfenato and sulfinato form. This work describes aspects of a particular nitrile hydratase, toyocamycin nitrile hydratase (TNH). Whereas most nitrile hydratases are heterodimeric, TNH is heterotrimeric, and yet what was discovered is that only the subunit containing the active site metal complex is required for activity. This single subunit analog of the protein was used for single turnover assays in ¹⁸O-labeled water to show with high resolution mass spectrometry that the source of the product amide oxygen is actually the enzyme itself and likely the sulfenato ligand oxygen acting as a nucleophile. The mechanism of the active site complex synthesis is described showing that this is self-catalytic in the presence of cobalt(II) and molecular oxygen. Advisors/Committee Members: Ghosh, Indraneel (advisor), Ghosh, Indraneel (committeemember), Bandarian, Vahe (committeemember), Cordes, Matthew (committeemember), Jewett, John (committeemember), Tomat, Elisa (committeemember).

Subjects/Keywords: Nitrile; Chemistry; Enzymology

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APA (6^th Edition):

Nelp, M. (2016). Biological Synthesis and Transformation of Nitriles . (Doctoral Dissertation). University of Arizona. Retrieved from http://hdl.handle.net/10150/621560

Chicago Manual of Style (16^th Edition):

Nelp, Micah. “Biological Synthesis and Transformation of Nitriles .” 2016. Doctoral Dissertation, University of Arizona. Accessed January 20, 2021. http://hdl.handle.net/10150/621560.

MLA Handbook (7^th Edition):

Nelp, Micah. “Biological Synthesis and Transformation of Nitriles .” 2016. Web. 20 Jan 2021.

Vancouver:

Nelp M. Biological Synthesis and Transformation of Nitriles . [Internet] [Doctoral dissertation]. University of Arizona; 2016. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/10150/621560.

Council of Science Editors:

Nelp M. Biological Synthesis and Transformation of Nitriles . [Doctoral Dissertation]. University of Arizona; 2016. Available from: http://hdl.handle.net/10150/621560

University of Utah

3. Bartholomew, Richard Mark. Isolation and metabolism of liver glycogen phosphorylase under normal and fasting dietary conditions;.

Degree: PhD, Biochemistry;, 1976, University of Utah

URL: http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/62/rec/735

► Phosphorylase and the enzymes which regulate its activity occur as complexes with glycogen in liver and can therefore be considered as compartmentalized enzymes. A study… (more)

▼ Phosphorylase and the enzymes which regulate its activity occur as complexes with glycogen in liver and can therefore be considered as compartmentalized enzymes. A study of the metabolism of glycogen phosphorylase was undertaken to determine whether or not this association with glycogen was an important factor in establishing the phosphorylase concentration at equilibrium. We also wanted to investigate if regulating phosphorylase levels supplemented the hormonal control of the enzyme's activity. Homogeneous glycogen phosphorylase b, free of phosphorylase a contamination, was isolated from rabbit liver by differential sedimentation of the glycogen-phosphorylase complex, digestion of the glycogen with a-amylase, and fractionation of the resulting sample by consecutive n-butyl Sepharose and DEAE cellulose chromatographies, followed by isoelectric focusing. Yields approaching 50 percent and 1600-fold purification were obtained, with resulting specific activities that were significantly higher than those expressed by the enzyme isolated by previous methods. Isolation of the enzyme in the absence of glycogen was accomplished by ethanol fractionation, adsorption of contaminating protein on calcium phosphate gel, ammonium sulfate precipitation, and slight modifications of the Sepharose, DEAE cellulose, and isoelectric focusing purification steps. After demonstrating that the isolated phosphorylase was representative of the total cellular content, its synthesis and degradation in fed and fasted animals was studied using both radioactive tracers and direct enzyme assay to monitor the turnover. A pulse of labeled leucine or lysine was injected into each of three rabbits. Incorporation of the label into phosphorylase and liver aldolase was simultaneously monitored by isolating the enzymes in homogeneous form at various times after injection and calculating their normalized specific radioactivilies. Results showed that phosphorylase was a more stable molecule, turning over at one-half to one-third the rate of aldolase. Approximations of half lives yielded values of 2-3 days for phosphorylase and slightly less than one day for aldolase. An independent approach to phosphorylase turnover was made possible through changes observed in the total content of the enzyme during adjustments to differing dietary conditions. Phosphorylase levels were determined directly in whole-liver homogenates after conversion to the b form at intervals during fasting and subsequent refeeding. On fasting, the total phosphorylase content decreased 63 percent within about four days to a new steady-state level and, upon refeeding, returned at a significantly slower rate to its normal concentration. It was established by isotope dilution analysis that the changes in phosphorylase activity were due to changes in the amount of enzyme present and not to alterations in its enzymatic activity. Therefore, significant changes in the rate constants for phosphorylase metabolism had occurred as a result of the fast. Analysis of the time approaches to end from the two…

Subjects/Keywords: Enzymology; Analysis

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APA (6^th Edition):

Bartholomew, R. M. (1976). Isolation and metabolism of liver glycogen phosphorylase under normal and fasting dietary conditions;. (Doctoral Dissertation). University of Utah. Retrieved from http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/62/rec/735

Chicago Manual of Style (16^th Edition):

Bartholomew, Richard Mark. “Isolation and metabolism of liver glycogen phosphorylase under normal and fasting dietary conditions;.” 1976. Doctoral Dissertation, University of Utah. Accessed January 20, 2021. http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/62/rec/735.

MLA Handbook (7^th Edition):

Bartholomew, Richard Mark. “Isolation and metabolism of liver glycogen phosphorylase under normal and fasting dietary conditions;.” 1976. Web. 20 Jan 2021.

Vancouver:

Bartholomew RM. Isolation and metabolism of liver glycogen phosphorylase under normal and fasting dietary conditions;. [Internet] [Doctoral dissertation]. University of Utah; 1976. [cited 2021 Jan 20]. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/62/rec/735.

Council of Science Editors:

Bartholomew RM. Isolation and metabolism of liver glycogen phosphorylase under normal and fasting dietary conditions;. [Doctoral Dissertation]. University of Utah; 1976. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/62/rec/735

Vanderbilt University

4. Sedgeman, Carl Andrew. Formation, Degradation, and Bypass of DNA-Protein Crosslinks.

Degree: PhD, Biochemistry, 2018, Vanderbilt University

URL: http://hdl.handle.net/1803/14171

► The preservation of DNA replication is requisite for cellular integrity and prevention of tumor formation and cell death. The Y-family DNA polymerases (Pol eta, kappa,… (more)

Subjects/Keywords: carcinogens; enzymology; DNA damage

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APA (6^th Edition):

Sedgeman, C. A. (2018). Formation, Degradation, and Bypass of DNA-Protein Crosslinks. (Doctoral Dissertation). Vanderbilt University. Retrieved from http://hdl.handle.net/1803/14171

Chicago Manual of Style (16^th Edition):

Sedgeman, Carl Andrew. “Formation, Degradation, and Bypass of DNA-Protein Crosslinks.” 2018. Doctoral Dissertation, Vanderbilt University. Accessed January 20, 2021. http://hdl.handle.net/1803/14171.

MLA Handbook (7^th Edition):

Sedgeman, Carl Andrew. “Formation, Degradation, and Bypass of DNA-Protein Crosslinks.” 2018. Web. 20 Jan 2021.

Vancouver:

Sedgeman CA. Formation, Degradation, and Bypass of DNA-Protein Crosslinks. [Internet] [Doctoral dissertation]. Vanderbilt University; 2018. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1803/14171.

Council of Science Editors:

Sedgeman CA. Formation, Degradation, and Bypass of DNA-Protein Crosslinks. [Doctoral Dissertation]. Vanderbilt University; 2018. Available from: http://hdl.handle.net/1803/14171

Vanderbilt University

5. Tomasiak, Thomas. Catalysis, inhibition, and signal transduction by menaquinol:fumarate oxidoreductase.

Degree: PhD, Pharmacology, 2011, Vanderbilt University

URL: http://hdl.handle.net/1803/10566

► Complex II superfamily members catalyze two separate reactions in respiration: interconversion of fumarate and succinate in the soluble milieu and interconversion of quinol and quinone… (more)

▼ Complex II superfamily members catalyze two separate reactions in respiration: interconversion of fumarate and succinate in the soluble milieu and interconversion of quinol and quinone in the membrane. Electrons liberated as a result of one reaction become substrates for the second, thermodynamically linking both sites and allowing complex II enzymes to catalyze electron entry to or exit from a respiratory chain. In anaerobic respiration, menaquinol:fumarate oxidoreductase (QFR) catalyzes the final step in the most widely used anaerobic respiratory pathway, fumarate reduction. This work analyzes structural and mechanistic details of function and inhibition at each of the two active sites. The first part examines transition state formation at the active site responsible for fumarate and succinate interconversion, the dicarboxylate site. It was found that two threonine residues, Thr-234 and Thr-244, might play important roles in attaining and stabilizing the transition state. The second part of this work focuses on details of dicarboxylate site inhibition by substrate-like molecules and of substrate activation. It was found that all tight binding ligands or ligands transformed by the enzyme also induce large optical shifts in an FAD cofactor and align an activatable bond along the active C4a-N5 axis of FAD. This bond overlap was proposed to play a part of an orbital steering mechanism to guide substrate and activate it for catalysis. This alignment was found be conserved in a number of flavoenzymes that catalyze dehydrogenation reactions, even with completely unrelated substrates and structural folds. The third part of this work focuses on the second active site, the quinone/quinol interconversion site. The E29L variant QFR was crystallized to study semiquinone formation. Preliminary results reveal that the substrate, menaquinone, may bind in a continuum of active site positions as the substrate undergoes catalysis. Another substrate, ubiquinone was shown to bring about the presence of strong electron density at a site located 13 angstroms away from menaquinone site. The forth and final part of this work examines the structural work done to isolate and stabilize complex formation between QFR and FliG, a part of the flagellar motor. Advisors/Committee Members: Tina Iverson (committee member), Heidi Hamm (committee member), Hassane Mchaourab (committee member), Jens Meiler (committee member), H. Alex Brown (Committee Chair).

Subjects/Keywords: respiration; membrane; crystallography; enzymology

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APA (6^th Edition):

Tomasiak, T. (2011). Catalysis, inhibition, and signal transduction by menaquinol:fumarate oxidoreductase. (Doctoral Dissertation). Vanderbilt University. Retrieved from http://hdl.handle.net/1803/10566

Chicago Manual of Style (16^th Edition):

Tomasiak, Thomas. “Catalysis, inhibition, and signal transduction by menaquinol:fumarate oxidoreductase.” 2011. Doctoral Dissertation, Vanderbilt University. Accessed January 20, 2021. http://hdl.handle.net/1803/10566.

MLA Handbook (7^th Edition):

Tomasiak, Thomas. “Catalysis, inhibition, and signal transduction by menaquinol:fumarate oxidoreductase.” 2011. Web. 20 Jan 2021.

Vancouver:

Tomasiak T. Catalysis, inhibition, and signal transduction by menaquinol:fumarate oxidoreductase. [Internet] [Doctoral dissertation]. Vanderbilt University; 2011. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1803/10566.

Council of Science Editors:

Tomasiak T. Catalysis, inhibition, and signal transduction by menaquinol:fumarate oxidoreductase. [Doctoral Dissertation]. Vanderbilt University; 2011. Available from: http://hdl.handle.net/1803/10566

University of Illinois – Urbana-Champaign

6. Ulrich, Emily C. Diverse enzyme reactions in phosphonate natural product biosynthesis.

Degree: PhD, Chemistry, 2018, University of Illinois – Urbana-Champaign

URL: http://hdl.handle.net/2142/101253

► A number of new biosynthetic gene clusters encoding phosphonate natural products have been characterized by mining of microbial genomes. Phosphonates, which are characterized by their… (more)

▼ A number of new biosynthetic gene clusters encoding phosphonate natural products have been characterized by mining of microbial genomes. Phosphonates, which are characterized by their hydrolytically stable carbon-phosphorus bond, are prevalent in everyday life, such as in commercially available herbicides and an antibiotic deemed critically important in the clinic. Their biosynthetic pathways are rich in novel chemistry, ranging from iron-dependent radical rearrangements to multiple methods of forming amide bonds. This thesis will include my contributions to understanding phosphonate enzymatic chemistry in more detail. Methylphosphonate (MPn) was recently isolated from marine surface waters and is proposed to be a precursor in the production of methane, which is supersaturated in these environments. The oxygenase MPn synthase (MPnS) produces MPn from 2-hydroxyethylphosphonate (2-HEP), a substrate also acted on by 2-HEP dioxygenase (HEPD) to make hydroxymethylphosphonate (HMP). Both enzymes are proposed to share a common mechanism up until the final intermediate consisting of an MPn radical and formate. Chapter 2 will discuss efforts to elucidate how each enzyme dictates individual product formation at this key branching step between the two mechanisms. An unprecedented 2-His-1-Gln facial triad, along with the identity of a second sphere residue, were determined to contribute to MPn versus HMP formation. This sequence motif was used to predict the prevalence of MPnS in other microbial genomes, leading to the functional characterization of an MPnS from the abundant marine bacterium Pelagibacter ubique. This discovery further supports MPn as a possible source of the supersaturated methane levels in the aerobic ocean. The tripeptide antibiotic dehydrophos consists of a phosphonate warhead attached to two proteinogenic amino acids which function to allow entry into target cells. During biosynthesis, these amino acids are transferred to the phosphonate through the action of aminoacyl-tRNA (aa-tRNA)-dependent ligases. Chapter 3 will discuss efforts to characterize the interaction of the enzyme DhpH-C with Leu-tRNALeu. Mutagenesis studies of both the enzyme and tRNA led to the conclusion that DhpH-C shares features of aa-tRNA recognition common to other aa-tRNA-dependent enzymes. In addition, DhpH-C was found to accept other amino acids for the production of multiple dipeptide phosphonates, although Leu-tRNALeu was the most efficient substrate with the fastest rate of product formation over time. These features of DhpH-C may aid in future studies to produce phosphonate analogs chemoenzymatically, and its modes of aa-tRNA substrate recognition appear to be applicable to the study of aa-tRNA-dependent enzymes in the production of different natural product classes. Biosynthesis of the clinically used antibiotic fosfomycin can occur by two convergent routes in Streptomyces and Pseudomonas species. The pathways only share the first and last steps, which are catalyzed by phosphoenolpyruvate mutase and… Advisors/Committee Members: van der Donk, Wilfred A. (advisor), van der Donk, Wilfred A. (Committee Chair), Martinis, Susan A. (committee member), Nair, Satish K. (committee member), Silverman, Scott K. (committee member).

Subjects/Keywords: Phosphonate; Natural product; Enzymology

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

APA (6^th Edition):

Ulrich, E. C. (2018). Diverse enzyme reactions in phosphonate natural product biosynthesis. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/101253

Chicago Manual of Style (16^th Edition):

Ulrich, Emily C. “Diverse enzyme reactions in phosphonate natural product biosynthesis.” 2018. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed January 20, 2021. http://hdl.handle.net/2142/101253.

MLA Handbook (7^th Edition):

Ulrich, Emily C. “Diverse enzyme reactions in phosphonate natural product biosynthesis.” 2018. Web. 20 Jan 2021.

Vancouver:

Ulrich EC. Diverse enzyme reactions in phosphonate natural product biosynthesis. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2018. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2142/101253.

Council of Science Editors:

Ulrich EC. Diverse enzyme reactions in phosphonate natural product biosynthesis. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2018. Available from: http://hdl.handle.net/2142/101253

McMaster University

7. Kelso, Jayne. Characterizing the mechanism and regulation of a rifamycin monooxygenase in Streptomyces venezuelae.

Degree: MSc, 2016, McMaster University

URL: http://hdl.handle.net/11375/20423

►

The rifamycins are a class of antibiotics which were once used almost exclusively to treat tuberculosis, but are currently receiving renewed interest. Resistance to rifamycins… (more)

▼

The rifamycins are a class of antibiotics which were once used almost exclusively to treat tuberculosis, but are currently receiving renewed interest. Resistance to rifamycins is most commonly attributed to mutations in the drug target, RNA polymerase. Yet environmental isolates are also able to enzymatically inactivate rifamycins in a number of ways. Recently, rifamycin resistance determinants from the environment were found to be closely associated with a so called rifamycin associated element (RAE). The region containing the RAE from an environmental strain was shown to induce gene expression in the presence of rifamycins, hinting at an inducible system for rifamycin resistance. In this work, we examine the RAE from a model organism for Streptomyces genetics, Streptomyces venezuelae. We confirm that the promoter region containing the RAE upstream of a rifamycin monooxygenase rox is inducible by rifamycins. The strains of S. venezuelae generated in this work can be used in future genetic studies on the RAE. As well, the rifamycin monooxygenase Rox was purified for the first time and characterized biochemically. The structure of Rox was obtained with and without the substrate rifampin. Steady state kinetics for the enzyme were determined with a number of substrates, and its ability to confer resistance to rifamycins was examined. Monooxygenated rifamycin SV compound was purified and structurally characterized by NMR analysis. We proposed an aromatic hydroxylase type mechanism for Rox, in which the enzyme hydroxylates the aromatic core of the rifamycin scaffold and causes a non-enzymatic C-N bond cleavage of the macrolactam ring. This is a new mechanism of rifamycin resistance, and sheds some light on the decomposition of rifamycins mediated by monooxygenation, which is still poorly understood.

Thesis

Master of Science (MSc)

Antibiotic resistance represents a major threat to global health. Infections that were once readily treatable are no longer so due to the rise in multidrug resistant bacteria. As our arsenal of effective antibiotics is depleted, new drugs are being discovered less and less frequently. This has caused the scientific community to get creative in coming up with treatments: trying combinations of antibiotics, using antibiotics which were once considered too toxic, and repurposing antibiotics for different bacteria. Rifamycins are a class of antibiotics most commonly used in the treatment of tuberculosis. However, they are becoming more widely used as a result of antibiotic resistance. There are a number of different ways bacteria can become resistant to the harmful effects of rifamycins: by modifying the target so the drug can no longer bind to it, actively pumping the drug out of the cell, or by changing the drug in some way so it is no longer effective. Bacteria in the environment use antibiotics as a form of chemical warfare to gain an advantage over their neighbours; therefore, they have had millions of years to evolve very effective methods of antibiotic resistance. By surveying…

Advisors/Committee Members: Wright, Gerard D, Biochemistry and Biomedical Sciences.

Subjects/Keywords: Antibiotic resistance; Enzymology; Rifamycins

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APA (6^th Edition):

Kelso, J. (2016). Characterizing the mechanism and regulation of a rifamycin monooxygenase in Streptomyces venezuelae. (Masters Thesis). McMaster University. Retrieved from http://hdl.handle.net/11375/20423

Chicago Manual of Style (16^th Edition):

Kelso, Jayne. “Characterizing the mechanism and regulation of a rifamycin monooxygenase in Streptomyces venezuelae.” 2016. Masters Thesis, McMaster University. Accessed January 20, 2021. http://hdl.handle.net/11375/20423.

MLA Handbook (7^th Edition):

Kelso, Jayne. “Characterizing the mechanism and regulation of a rifamycin monooxygenase in Streptomyces venezuelae.” 2016. Web. 20 Jan 2021.

Vancouver:

Kelso J. Characterizing the mechanism and regulation of a rifamycin monooxygenase in Streptomyces venezuelae. [Internet] [Masters thesis]. McMaster University; 2016. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/11375/20423.

Council of Science Editors:

Kelso J. Characterizing the mechanism and regulation of a rifamycin monooxygenase in Streptomyces venezuelae. [Masters Thesis]. McMaster University; 2016. Available from: http://hdl.handle.net/11375/20423

McMaster University

8. Sapiano, Matthew J. LIPID A REGIOSELECTIVITY OF THE ESCHERICHIA COLI PALMITOYLTRANSFERASE PAGP.

Degree: 2014, McMaster University

URL: http://hdl.handle.net/11375/16317

►

The outer membrane palmitoyltransferase PagP possesses regioselectivity for the palmitoylation of the (R)-3-hydroxymyristate chain at position 2 on the proximal glucosamine unit of lipid A.… (more)

Subjects/Keywords: Biochemistry; Membrane; Enzymology; Microbiology

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APA (6^th Edition):

Sapiano, M. J. (2014). LIPID A REGIOSELECTIVITY OF THE ESCHERICHIA COLI PALMITOYLTRANSFERASE PAGP. (Thesis). McMaster University. Retrieved from http://hdl.handle.net/11375/16317

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Sapiano, Matthew J. “LIPID A REGIOSELECTIVITY OF THE ESCHERICHIA COLI PALMITOYLTRANSFERASE PAGP.” 2014. Thesis, McMaster University. Accessed January 20, 2021. http://hdl.handle.net/11375/16317.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Sapiano, Matthew J. “LIPID A REGIOSELECTIVITY OF THE ESCHERICHIA COLI PALMITOYLTRANSFERASE PAGP.” 2014. Web. 20 Jan 2021.

Vancouver:

Sapiano MJ. LIPID A REGIOSELECTIVITY OF THE ESCHERICHIA COLI PALMITOYLTRANSFERASE PAGP. [Internet] [Thesis]. McMaster University; 2014. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/11375/16317.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Sapiano MJ. LIPID A REGIOSELECTIVITY OF THE ESCHERICHIA COLI PALMITOYLTRANSFERASE PAGP. [Thesis]. McMaster University; 2014. Available from: http://hdl.handle.net/11375/16317

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Oregon State University

9. Diaz, David D. Carbon cycling and priming of soil organic matter decomposition in a forest soil following glucose additions.

Degree: MS, Soil Science, 2008, Oregon State University

URL: http://hdl.handle.net/1957/8620

► Soils are a globally significant carbon (C) pool and have the potential to respond to elevated CO2 and environmental changes through positive feedback cycles that… (more)

▼ Soils are a globally significant carbon (C) pool and have the potential to respond to elevated CO2 and environmental changes through positive feedback cycles that enhance the turnover of soil organic matter (SOM). Understanding the mechanisms governing the turnover of SOM is particularly important for modeling the fate of C in soils under predicted environmental changes. The change in turnover of SOM following additions of labile C through natural root and litter deposition as well as from human activities such as fertilizer application is known as priming, and the mechanisms governing this process are poorly understood but may be important components determining soil C balance. Many studies have utilized experimental additions of labile C and variously interpreted the primed C to derive from the activation and turnover of microbial biomass or from the increased decomposition of non-microbial SOM. The objective of this study was to evaluate changes in the activities of SOM-degrading enzymes as a potential mechanism for observed priming effects and to determine whether the priming response was related to the availability of “primable” C from root and litter inputs to soil. 13C-labeled glucose was injected in the field into surface soils of an old-growth coniferous forest on the western side of the central Oregon Cascade Range. The fate of these small glucose additions (100 µg C g-1 dry soil) was traced into soil C pools and captured as respired CO2 over the course of a 22-day experiment. The forest soils considered in this study have undergone ten years of selective exclusion of root and/or litter inputs as part of a larger experiment known as the Detrital Input and Removal Treatments (DIRT). Plots without roots showed negative priming responses to glucose additions with reduced turnover of SOM due to a preferential switch from SOM to glucose as a C source and through an apparent generalized suppression of microbial activity. Positive priming was observed in soils receiving regular C inputs from roots and litter. Both positive and negative priming were observed in plots with litter inputs excluded depending upon which method of priming quantification was used. In soils with C-input limitations (i.e., No Roots, No Litter, No Inputs), the magnitude and direction of priming was negatively related to background respiration rates with highly active soils in these plots showing the strongest negative priming effects. Control soils showed no relationship between background respiration rates and priming effects. The potential activities of β-glucosidase, phenol oxidase, and peroxidase showed no consistent relationship to glucose additions or priming effects and suggest that the production of enzymes in response to labile C inputs may not be the controlling mechanism for priming in these soils. The amount of primed C mineralized throughout the course of the experiment is consistent with earlier hypotheses that the activation and increased turnover of microbial biomass C is the primary source of primed C. … Advisors/Committee Members: Myrold, David D. (advisor), Kleber, Markus (committee member).

Subjects/Keywords: carbon cycling; Soil enzymology – Oregon

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APA (6^th Edition):

Diaz, D. D. (2008). Carbon cycling and priming of soil organic matter decomposition in a forest soil following glucose additions. (Masters Thesis). Oregon State University. Retrieved from http://hdl.handle.net/1957/8620

Chicago Manual of Style (16^th Edition):

Diaz, David D. “Carbon cycling and priming of soil organic matter decomposition in a forest soil following glucose additions.” 2008. Masters Thesis, Oregon State University. Accessed January 20, 2021. http://hdl.handle.net/1957/8620.

MLA Handbook (7^th Edition):

Diaz, David D. “Carbon cycling and priming of soil organic matter decomposition in a forest soil following glucose additions.” 2008. Web. 20 Jan 2021.

Vancouver:

Diaz DD. Carbon cycling and priming of soil organic matter decomposition in a forest soil following glucose additions. [Internet] [Masters thesis]. Oregon State University; 2008. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1957/8620.

Council of Science Editors:

Diaz DD. Carbon cycling and priming of soil organic matter decomposition in a forest soil following glucose additions. [Masters Thesis]. Oregon State University; 2008. Available from: http://hdl.handle.net/1957/8620

Virginia Tech

10. Gagliano, Elisa. A Bioinformatics Approach to Identifying Radical SAM (S-Adenosyl-L-Methionine) Enzymes.

Degree: MSin Life Sciences, Biochemistry, 2020, Virginia Tech

URL: http://hdl.handle.net/10919/98736

► Radical SAM enzymes are ancient, essential enzymes that perform chemical reactions in virtually all living organisms. We do know that they are involved in producing… (more)

Subjects/Keywords: Bioinformatics; Radical Biochemistry; Enzymology

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APA (6^th Edition):

Gagliano, E. (2020). A Bioinformatics Approach to Identifying Radical SAM (S-Adenosyl-L-Methionine) Enzymes. (Masters Thesis). Virginia Tech. Retrieved from http://hdl.handle.net/10919/98736

Chicago Manual of Style (16^th Edition):

Gagliano, Elisa. “A Bioinformatics Approach to Identifying Radical SAM (S-Adenosyl-L-Methionine) Enzymes.” 2020. Masters Thesis, Virginia Tech. Accessed January 20, 2021. http://hdl.handle.net/10919/98736.

MLA Handbook (7^th Edition):

Gagliano, Elisa. “A Bioinformatics Approach to Identifying Radical SAM (S-Adenosyl-L-Methionine) Enzymes.” 2020. Web. 20 Jan 2021.

Vancouver:

Gagliano E. A Bioinformatics Approach to Identifying Radical SAM (S-Adenosyl-L-Methionine) Enzymes. [Internet] [Masters thesis]. Virginia Tech; 2020. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/10919/98736.

Council of Science Editors:

Gagliano E. A Bioinformatics Approach to Identifying Radical SAM (S-Adenosyl-L-Methionine) Enzymes. [Masters Thesis]. Virginia Tech; 2020. Available from: http://hdl.handle.net/10919/98736

Princeton University

11. Jani, Krupa Shashank. Mechanistic studies of epigenetic regulation by the H3 tail .

Degree: PhD, 2019, Princeton University

URL: http://arks.princeton.edu/ark:/88435/dsp01js956j618

► The N-terminal tail of histone H3 is heavily post-translationally modified. Numerous regulatory lysine residues are methylated, and these modifications play critical roles in the regulation… (more)

▼ The N-terminal tail of histone H3 is heavily post-translationally modified. Numerous regulatory lysine residues are methylated, and these modifications play critical roles in the regulation of gene expression. Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of genes important in growth and development. Intriguingly, H3K27me3 is mutually exclusive with H3K36 tri-methylation on the same histone tail. Disruptions in this crosstalk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of this crosstalk are unclear. This thesis presents work revealing an unusual mode of allosteric enzyme modulation by its histone H3 substrate molecule. PRC2-mediated H3K27 methylation is sensitive to the chromatin environment, including the methylation state of H3K36 on the same histone tail. We demonstrate that PRC2 is allosterically activated in cis by the unmodified side chain of H3K36, and that this activation results in a 5-fold increase in the kcat of its enzymatic activity catalyzing H3K27 methylation compared to activity on a substrate methylated at H3K36. Utilizing a photocrosslinking-MS strategy and histone methyltransferase activity assays with PRC2 mutants, we find that the catalytic subunit EZH2 contains a specific sensing pocket for H3K36 methylation state which allows the PRC2 complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. Avenues for inhibition of PRC2 by targeting its interactions with the H3 tail are also explored. Beyond its interactions with polycomb machinery, H3K36 and other sections of the H3 tail may also have roles of epigenetic regulation of other cellular processes such as DNA damage response. Advisors/Committee Members: Muir, Tom W (advisor).

Subjects/Keywords: Enzymology; Epigenetics; H3; PRC2

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APA (6^th Edition):

Jani, K. S. (2019). Mechanistic studies of epigenetic regulation by the H3 tail . (Doctoral Dissertation). Princeton University. Retrieved from http://arks.princeton.edu/ark:/88435/dsp01js956j618

Chicago Manual of Style (16^th Edition):

Jani, Krupa Shashank. “Mechanistic studies of epigenetic regulation by the H3 tail .” 2019. Doctoral Dissertation, Princeton University. Accessed January 20, 2021. http://arks.princeton.edu/ark:/88435/dsp01js956j618.

MLA Handbook (7^th Edition):

Jani, Krupa Shashank. “Mechanistic studies of epigenetic regulation by the H3 tail .” 2019. Web. 20 Jan 2021.

Vancouver:

Jani KS. Mechanistic studies of epigenetic regulation by the H3 tail . [Internet] [Doctoral dissertation]. Princeton University; 2019. [cited 2021 Jan 20]. Available from: http://arks.princeton.edu/ark:/88435/dsp01js956j618.

Council of Science Editors:

Jani KS. Mechanistic studies of epigenetic regulation by the H3 tail . [Doctoral Dissertation]. Princeton University; 2019. Available from: http://arks.princeton.edu/ark:/88435/dsp01js956j618

Montana State University

12. Boswell, Nicholas William Bradford. Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate.

Degree: MS, College of Letters & Science, 2011, Montana State University

URL: https://scholarworks.montana.edu/xmlui/handle/1/955

► Hydrogenases catalyze the reversible reduction of protons using complex metal clusters with unusual ligands. The catalytic center of the [FeFe]-hydrogenases is called the H-cluster, and… (more)

▼ Hydrogenases catalyze the reversible reduction of protons using complex metal clusters with unusual ligands. The catalytic center of the [FeFe]-hydrogenases is called the H-cluster, and is characterized by a [4Fe-4S] cluster connected via a cysteine thiolate to a 2Fe subcluster coordinated by carbon monoxide and cyanide ligands as well as a bridging dithiolate. Assembly of the H-cluster is carried out by three hydrogenase maturation proteins: HydE, HydF, and HydG. HydF is a GTPase and has been implicated to serve as a scaffold for assembly of the 2Fe subcluster of the H-cluster. HydE and HydG are radical S-adenosylmethionine (SAM) enzymes and thus are thought to utilize reductive cleavage of SAM to initiate radical chemistry. HydG has been shown to catalyze the formation of the carbon monoxide and cyanide ligands of the H-cluster utilizing tyrosine as a substrate. HydE, therefore, has been proposed to be responsible for biosynthesis of the dithiolate ligand of the H-cluster. The aim of this study was to biochemically characterize active, Fe-S reconstituted HydE and to identify the substrate of this radical SAM enzyme. Questions to be studied also included studying the role of HydE in H-cluster maturation. This study used protein purified from recombinant E. coli. The purified protein was chemically reconstituted with iron and sulfide, and used for spectroscopic characterization and HPLC based activity assays. Colorimetric assays were also used for protein characterization and to test for the consumption of substrate. The results indicate that cysteine is likely the substrate of HydE. Activity assays show that HydE- catalyzed SAM cleavage is stimulated in the presence of cysteine, and HydF purified from different genetic backgrounds shows a spectroscopic shift in the lambda max when both HydE and cysteine are present during growth. Spectroscopic characterization confirms that HydE is an Fe-S containing radical SAM enzyme and that cysteine may be a substrate during [FeFe]-hydrogenase H-cluster maturation. Advisors/Committee Members: Chairperson, Graduate Committee: Joan B. Broderick (advisor).

Subjects/Keywords: Hydrogenase.; Biochemistry.; Enzymology.; Fuel.

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APA (6^th Edition):

Boswell, N. W. B. (2011). Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate. (Masters Thesis). Montana State University. Retrieved from https://scholarworks.montana.edu/xmlui/handle/1/955

Chicago Manual of Style (16^th Edition):

Boswell, Nicholas William Bradford. “Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate.” 2011. Masters Thesis, Montana State University. Accessed January 20, 2021. https://scholarworks.montana.edu/xmlui/handle/1/955.

MLA Handbook (7^th Edition):

Boswell, Nicholas William Bradford. “Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate.” 2011. Web. 20 Jan 2021.

Vancouver:

Boswell NWB. Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate. [Internet] [Masters thesis]. Montana State University; 2011. [cited 2021 Jan 20]. Available from: https://scholarworks.montana.edu/xmlui/handle/1/955.

Council of Science Editors:

Boswell NWB. Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate. [Masters Thesis]. Montana State University; 2011. Available from: https://scholarworks.montana.edu/xmlui/handle/1/955

University of Illinois – Urbana-Champaign

13. Aubourg, Nadine. Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT).

Degree: MS, Food Science & Human Nutrition, 2015, University of Illinois – Urbana-Champaign

URL: http://hdl.handle.net/2142/78799

► Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine to form dimethylglycine and methionine, respectively. BMHT is primarily expressed in… (more)

▼ Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine to form dimethylglycine and methionine, respectively. BMHT is primarily expressed in the liver and kidney of mammals. BHMT catalyzes an ordered bi bi reaction where the first product released, dimethylglycine, can compete for the betaine binding site and inhibit homocysteine utilization. There is considerable interest in the regulation of homocysteine metabolism since even moderate elevations in plasma total homocysteine have been established as an independent risk factor for the development of vascular diseases and thrombosis. In people with homocystinuria, treatment using supplemental betaine showed a significant decrease in total plasma homocysteine but does not lower homocysteine levels within the normal range, and the moderate levels that remain are highly correlated with vascular disease. Therefore, the BHMT catalyzed reaction is a target for the treatment of homocystinuria. Finding alternative methyl donors for the BHMT reaction that following methyl transfer have less potent inhibitory properties than dimethylglycine are desired. Sulfonium analogs of betaine are considered for this research. The main objectives of this research were to determine the inhibitory properties of the demethylated product of betaine and its sulfonium analogs and also to determine the Michaelis constants in order to use them as alternative methyl donors for the BHMT reaction in homocystinuric patients. The methyl donor substrates used are betaine, dimethylsulfonioacetate, dimethylsulfoniopropionate, and their respective demethylated products are dimethylglycine, methylthioacetate and methylpropionate. Dimethylglycine had the lowest IC50 values, ranging from 28 to 35 µM for all three enzymes. Methylthioacetate had IC50 values ranging from 65 to 106 µM, and values for methylthiopropionate ranged from 400 to 800 µM. There was no significant difference between the IC50 values obtained for the different enzymes when assayed in the presence of DMG or MTA, but for MTP the IC50 values were significantly different from one enzyme to another. Kinetic studies for betaine was conducted for all three enzymes. The Km of betaine varied from 1.8±0.5 mM, 0.7±0.06, and 0.5±0.06 mM for overexpressed human BHMT, pig and rat liver BHMT, respectively. We are unable to determine the Km for DMSA and DMSP. Further studies as the catalytic efficient (Kcat) for all three substrates are needed in other to determine which one could be a better alternative treatment for homocystinuria.

Subjects/Keywords: Homocysteine; Methionine; Betaine; Enzymology; Kinetic

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APA (6^th Edition):

Aubourg, N. (2015). Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT). (Thesis). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/78799

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Aubourg, Nadine. “Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT).” 2015. Thesis, University of Illinois – Urbana-Champaign. Accessed January 20, 2021. http://hdl.handle.net/2142/78799.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Aubourg, Nadine. “Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT).” 2015. Web. 20 Jan 2021.

Vancouver:

Aubourg N. Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT). [Internet] [Thesis]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2142/78799.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Aubourg N. Kinetic characterization of methyl donor substrates and inhibitors of human, pig, and rat liver betaine homocysteine S-methyltransferase (BHMT). [Thesis]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/78799

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Arizona

14. McCarty, Reid Michael. Elucidation of the Biosynthetic Pathway for 7-Deazapurines .

Degree: 2011, University of Arizona

URL: http://hdl.handle.net/10150/204334

► Small molecules containing a 7-deazapurine moiety are ubiquitous in nature. They comprise a broad range of structurally diverse antibiotics produced by terrestrial and marine microorganisms… (more)

Subjects/Keywords: antibiotics; enzymology; natural products; tRNA

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APA (6^th Edition):

McCarty, R. M. (2011). Elucidation of the Biosynthetic Pathway for 7-Deazapurines . (Doctoral Dissertation). University of Arizona. Retrieved from http://hdl.handle.net/10150/204334

Chicago Manual of Style (16^th Edition):

McCarty, Reid Michael. “Elucidation of the Biosynthetic Pathway for 7-Deazapurines .” 2011. Doctoral Dissertation, University of Arizona. Accessed January 20, 2021. http://hdl.handle.net/10150/204334.

MLA Handbook (7^th Edition):

McCarty, Reid Michael. “Elucidation of the Biosynthetic Pathway for 7-Deazapurines .” 2011. Web. 20 Jan 2021.

Vancouver:

McCarty RM. Elucidation of the Biosynthetic Pathway for 7-Deazapurines . [Internet] [Doctoral dissertation]. University of Arizona; 2011. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/10150/204334.

Council of Science Editors:

McCarty RM. Elucidation of the Biosynthetic Pathway for 7-Deazapurines . [Doctoral Dissertation]. University of Arizona; 2011. Available from: http://hdl.handle.net/10150/204334

Universitat de Valencia

15. Cabrera Luque, Juan Manuel. Discovery of novel pathways of microbial arginine biosynthesis .

Degree: 2012, Universitat de Valencia

URL: http://hdl.handle.net/10550/23189

► The amino acid L-arginine is an essential component of all living organisms. Its importance resides in the variety of functions that arginine itself, along with… (more)

▼ The amino acid L-arginine is an essential component of all living organisms. Its importance resides in the variety of functions that arginine itself, along with some intermediary metabolites involved in its de novo synthesis in the cell. In many prokaryotes, fungi, and plants the de novo biosynthesis of arginine proceeds from glutamate in eight enzymatic steps (Figure 2). The first committed step of this pathway is the N-acetylation of glutamate. Acetylation of the early precursors of arginine distinguishes them from the analogous intermediates in the biosynthesis of proline. Although each and every step of the pathway is essential for its completion, transcarbamylation of ornithine to produce citrulline is one of the crucial steps in the pathway. This reaction is catalyzed by the enzyme ornithine transcarbamylase (OTC). The transcarbamylases family of enzymes belongs to the carboxyl- and carbamoyltransferases group, EC 2.1.3, a family that comprises enzymes that catalyze the transfer of a carbamoyl group from carbamylphosphate (CP) to an amino or oxygen group of a second substrate. Members of the transcarbamylase family can be identified based on sequence identities in the N-terminal or CP-binding domain as all the members of this family share common residues involved in the binding of CP to the enzyme. All the evolutionary conserved motifs present in the transcarbamylase family have led to the erroneous annotation of a large, yet increasing, number of genes as OTCases without experimental confirmation of such activity. The case of the transcarbamylase-like gene found in B. fragilis illustrates this statement. The present thesis work is focused in the case of two families of misannotated microbial transcarbamylases that have been identified as essential for arginine biosynthesis, but which lacked the ability to catalyze the conversion of ornithine into citrulline. The objectives of the present thesis work are the biochemical and molecular characterization of the N-acetylornithine transcarbamylase (AOTC) activity of XcArgF’ as well as the elucidation of the enzymatic activity of BfArgF’ and its biochemical and molecular characterization. We show that Xanthomonads and Bacteroidetes use novel pathways for the de novo arginine biosynthesis mediated by acetyl- and succinyl-ornithine transcarbamylases, we identify a recognition motif for these enzymes and we provide evidence supporting the view that the existence of succinyl-ornithine transcarbamylase requires that the first five steps of the arginine biosynthesis pathway use succinylated rather than acetylated intermediates. Advisors/Committee Members: Morizono, Hiroki (advisor).

Subjects/Keywords: Arginine; Biosynthesis; Enzymology; Kinetics

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APA (6^th Edition):

Cabrera Luque, J. M. (2012). Discovery of novel pathways of microbial arginine biosynthesis . (Doctoral Dissertation). Universitat de Valencia. Retrieved from http://hdl.handle.net/10550/23189

Chicago Manual of Style (16^th Edition):

Cabrera Luque, Juan Manuel. “Discovery of novel pathways of microbial arginine biosynthesis .” 2012. Doctoral Dissertation, Universitat de Valencia. Accessed January 20, 2021. http://hdl.handle.net/10550/23189.

MLA Handbook (7^th Edition):

Cabrera Luque, Juan Manuel. “Discovery of novel pathways of microbial arginine biosynthesis .” 2012. Web. 20 Jan 2021.

Vancouver:

Cabrera Luque JM. Discovery of novel pathways of microbial arginine biosynthesis . [Internet] [Doctoral dissertation]. Universitat de Valencia; 2012. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/10550/23189.

Council of Science Editors:

Cabrera Luque JM. Discovery of novel pathways of microbial arginine biosynthesis . [Doctoral Dissertation]. Universitat de Valencia; 2012. Available from: http://hdl.handle.net/10550/23189

University of Guelph

16. Mallette, Evan. Structural and functional characterization of the aminoacetone utilization microcompartment from Mycobacterium smegmatis MC2 155.

Degree: PhD, Department of Molecular and Cellular Biology, 2019, University of Guelph

URL: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/16112

► Bacterial microcompartments are proteinacious complexes made by bacteria, which metabolize volatile or cytotoxic chemicals, by sequestering a series of reactions within a selectively permeable shell.… (more)

▼ Bacterial microcompartments are proteinacious complexes made by bacteria, which metabolize volatile or cytotoxic chemicals, by sequestering a series of reactions within a selectively permeable shell. This thesis focused on characterizing the catalytic functions and thus the metabolism of an aminoacetone utilization microcompartment (AAUM) found in Rhodococcus and Mycobacterium species, using the prototypical operon found in Mycobacterium smegmatis MC2 155 as a subject for investigation. Of the four enzymes associated with the AAUM, catalytic functions and structures were determined for two of the enzymes from M. smegmatis and for a homolog of a third enzyme. The first enzyme characterized was a stereospecific alcohol dehydrogenase catalyzing the reduction of 1-amino-2-propanone forming S-(+)-1-amino-2-propanol. The second enzyme was characterized as a 1-amino-2-propanol O-kinase with a preference for the S-isomer, able to also phosphorylate the R-isomer and amino-alcohols of varying lengths. An ortholog of the AAUM associated class-III aminotransferase from Mesorhizobium loti was characterized as a phosphopropanolamine phospholyase, forming ammonia, inorganic phosphate, and propionaldehyde. The remaining enzyme from the AAUM is annotated as a coenzyme A acylating aldehyde dehydrogenase, proposed to acylate coenzyme A with propionaldehyde produced by the phospholyase enzyme resulting in propionyl-CoA production. From the determined enzyme functions, I propose a metabolic pathway for the AAUM, converting aminoacetone to propionyl-CoA for use in central metabolism. The structures of the four shell proteins from the AAUM were determined by X-ray crystallography; proposed to form an icosahedral shell, with hexagonal oligomers forming the facets capped at the apices by pentagonal oligomers. The hexagonal hexameric bacterial microcompartment shell protein (BMC-H) formed a negatively charged pore, proposed to function in transport of a positively charged molecule across the shell. Both hexagonal trimeric bacterial microcompartment shell proteins (BMC-Ts) formed stacked trimeric rings with loops forming a tight interface at the center of each ring, proposed to undergo conformational shifts to open a large diameter pore for intermittent large molecule exchange. The pentagonal pentameric bacterial microcompartment shell protein (BMC-P) formed a roughly pyramidal oligomer proposed to fulfill an apex capping function. Using current modeling techniques, we were able to propose an architecture for the microcompartment shell. Advisors/Committee Members: Kimber, Matthew (advisor).

Subjects/Keywords: microcompartment; crystallography; mycobacterium; enzymology

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APA (6^th Edition):

Mallette, E. (2019). Structural and functional characterization of the aminoacetone utilization microcompartment from Mycobacterium smegmatis MC2 155. (Doctoral Dissertation). University of Guelph. Retrieved from https://atrium.lib.uoguelph.ca/xmlui/handle/10214/16112

Chicago Manual of Style (16^th Edition):

Mallette, Evan. “Structural and functional characterization of the aminoacetone utilization microcompartment from Mycobacterium smegmatis MC2 155.” 2019. Doctoral Dissertation, University of Guelph. Accessed January 20, 2021. https://atrium.lib.uoguelph.ca/xmlui/handle/10214/16112.

MLA Handbook (7^th Edition):

Mallette, Evan. “Structural and functional characterization of the aminoacetone utilization microcompartment from Mycobacterium smegmatis MC2 155.” 2019. Web. 20 Jan 2021.

Vancouver:

Mallette E. Structural and functional characterization of the aminoacetone utilization microcompartment from Mycobacterium smegmatis MC2 155. [Internet] [Doctoral dissertation]. University of Guelph; 2019. [cited 2021 Jan 20]. Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/16112.

Council of Science Editors:

Mallette E. Structural and functional characterization of the aminoacetone utilization microcompartment from Mycobacterium smegmatis MC2 155. [Doctoral Dissertation]. University of Guelph; 2019. Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/16112

17. Lin, Yi. Structural and Functional Studies of Urea Amidolyase.

Degree: 2014, Marquette University

URL: https://epublications.marquette.edu/dissertations_mu/381

► Urea amidolyase (UAL) is a key virulence factor that regulates the yeast to hyphae switch in the opportunistic pathogen, Candida albicans. UAL is a multi-domain… (more)

▼ Urea amidolyase (UAL) is a key virulence factor that regulates the yeast to hyphae switch in the opportunistic pathogen, Candida albicans. UAL is a multi-domain enzyme with two enzyme activities: urea carboxylase (UC) and allophanate hydrolase (AH). UC is a biotin-dependent carboxylase that adds a carboxyl group to urea to make allophanate by the coordinated action of three domains. Allophanate is subsequently hydrolyzed into NH3 and CO2 in the AH domain. Studies on the structure and function of UAL may lead to treatments for systemic candidiasis and can serve to clarify the molecular basis for multi-functional swinging arm enzymes. In the present study, the first structure of AH was solved by X-ray crystallography. Site-directed mutagenesis and steady-state kinetic analysis of Granulibacter bethesdensis AH reveal a role for two residues, Tyr299and Arg307, in maintaining substrate stringency and providing transition state stabilization. In addition, as UAL activity is essential for urea-dependent growth of yeast, a yeast genetic screen was developed to identify key functional residues in UAL. Random mutations were introduced in the targeted region of UAL, and the roles of an active site loop and distant residues in catalysis were highlighted in AH. The mechanisms of intermediate transfer between UC and AH were also studied. To investigate whether allophanate is channeled between the active sites, co-purification studies and substrate channeling assays were performed in vitro to detect stable or transient interactions between UC and AH. No strong coupling was detected between UC and AH. In addition, the coupling efficiency between the individual catalytic domains in UC is low, indicating that an as yet undiscovered activator may serve to facilitate the coordination among the three catalytic domains of UC. Taken together, these studies describe UAL as a complex, multi-functional enzyme that exhibits a high degree of substrate specificity in each domain, but does not require efficient substrate channeling to accomplish catalysis. These descriptions serve to advance the understanding of the mechanism of multi-functional enzymes as a whole. Advisors/Committee Members: St. Maurice, Martin, Fu, Jianhua, Stuart, Rosemary.

Subjects/Keywords: Enzymology; Genetics; Structural biology; Biology

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APA (6^th Edition):

Lin, Y. (2014). Structural and Functional Studies of Urea Amidolyase. (Thesis). Marquette University. Retrieved from https://epublications.marquette.edu/dissertations_mu/381

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Lin, Yi. “Structural and Functional Studies of Urea Amidolyase.” 2014. Thesis, Marquette University. Accessed January 20, 2021. https://epublications.marquette.edu/dissertations_mu/381.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Lin, Yi. “Structural and Functional Studies of Urea Amidolyase.” 2014. Web. 20 Jan 2021.

Vancouver:

Lin Y. Structural and Functional Studies of Urea Amidolyase. [Internet] [Thesis]. Marquette University; 2014. [cited 2021 Jan 20]. Available from: https://epublications.marquette.edu/dissertations_mu/381.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Lin Y. Structural and Functional Studies of Urea Amidolyase. [Thesis]. Marquette University; 2014. Available from: https://epublications.marquette.edu/dissertations_mu/381

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Manchester

18. White, Mark. Biochemical Characterisation of a Novel Decarboxylase System.

Degree: 2015, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:269228

► The Fdc1 and Pad1 decarboxylase system from Saccharomyces cerevisiae has been identified as a potential candidate to feature in novel biofuel production pathways based on… (more)

▼ The Fdc1 and Pad1 decarboxylase system from Saccharomyces cerevisiae has been identified as a potential candidate to feature in novel biofuel production pathways based on its ability to catalyze the transformation of sorbic acid, an antimicrobial compound, to 1, 3-pentadiene, a volatile hydrocarbon. Although information on the system is currently insufficient to permit a full assessment of its potential for future commercialization, it is hoped that (rational) engineering approaches can be used to evolve the enzymes to produce more desirable hydrocarbons. This requires biochemical characterization of the proteins. Genetic manipulation experiments have indicated that both enzymes are required for activity. However, no in vitro studies were conducted to verify the function, determine the relationship or establish the cofactor requirements of Fdc1 and Pad1. Results reported here establish that Fdc1 is the enzyme responsible for catalyzing decarboxylation, requiring a novel cofactor synthesized by Pad1 (or the bacterial homologue UbiX) for activity. High resolution crystal structures and mass spectrometry data from Fdc1 co-expressed with UbiX have indicated that the cofactor corresponds to a modified flavin mononucleotide (FMN) that has been extended with a C5-unit through linkages at the N5 and C6 atoms, creating a fourth, non-aromatic ring on the isoalloxazine group. Subsequent solution studies have established that this modification is achieved through isoprene chemistry, with UbiX facilitating prenyl transfer from dimethylallyl monophosphate (DMAP) to FMN. Analysis of wild type and mutant UbiX constructs by kinetic X-ray crystallography has allowed several distinct stages of the prenyl transfer reaction to be trapped, establishing that the protein uses a number of chemical strategies similar to terpene synthases to generate its product. The active site is dominated by pi systems, which aid heterolytic cleavage of the isoprene precursors phosphate-C1’ bond following FMN reduction, leading to the formation of an N5-C1’ intermediate. UbiX then acts as a chaperone for adduct reorientation, potentially via a transient tertiary carbocation, ultimately resulting in ring closure between the C6 and C3’. This work has established the biochemical principles underpinning the Fdc1 and Pad/UbiX decarboxylase system, providing a platform from which rational evolution approaches can be applied to the enzymes, specifically Fdc1, to improve their validity in the biofuels industry. It has also identified a novel cofactor that extends the previously well-documented flavin and isoprenoid repertoire. Advisors/Committee Members: SCRUTTON, NIGEL NS, Leys, David, Scrutton, Nigel.

Subjects/Keywords: Decarboxylation; Flavin; Crystallography; Prenyltransfer; Enzymology

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APA (6^th Edition):

White, M. (2015). Biochemical Characterisation of a Novel Decarboxylase System. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:269228

Chicago Manual of Style (16^th Edition):

White, Mark. “Biochemical Characterisation of a Novel Decarboxylase System.” 2015. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:269228.

MLA Handbook (7^th Edition):

White, Mark. “Biochemical Characterisation of a Novel Decarboxylase System.” 2015. Web. 20 Jan 2021.

Vancouver:

White M. Biochemical Characterisation of a Novel Decarboxylase System. [Internet] [Doctoral dissertation]. University of Manchester; 2015. [cited 2021 Jan 20]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:269228.

Council of Science Editors:

White M. Biochemical Characterisation of a Novel Decarboxylase System. [Doctoral Dissertation]. University of Manchester; 2015. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:269228

University of Georgia

19. Lamattina, Joseph William. Discovery and characterization of a novel heme-iron acquisition pathway from enterohemorrhagic e. coli.

Degree: 2017, University of Georgia

URL: http://hdl.handle.net/10724/36798

► Iron is an essential nutrient bacteria must acquire to survive within their niche, which can be a major barrier during colonization and pathogenesis. Therefore, pathogenic… (more)

Subjects/Keywords: Heme degradation; enzymology; radical SAM

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APA (6^th Edition):

Lamattina, J. W. (2017). Discovery and characterization of a novel heme-iron acquisition pathway from enterohemorrhagic e. coli. (Thesis). University of Georgia. Retrieved from http://hdl.handle.net/10724/36798

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Lamattina, Joseph William. “Discovery and characterization of a novel heme-iron acquisition pathway from enterohemorrhagic e. coli.” 2017. Thesis, University of Georgia. Accessed January 20, 2021. http://hdl.handle.net/10724/36798.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Lamattina, Joseph William. “Discovery and characterization of a novel heme-iron acquisition pathway from enterohemorrhagic e. coli.” 2017. Web. 20 Jan 2021.

Vancouver:

Lamattina JW. Discovery and characterization of a novel heme-iron acquisition pathway from enterohemorrhagic e. coli. [Internet] [Thesis]. University of Georgia; 2017. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/10724/36798.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Lamattina JW. Discovery and characterization of a novel heme-iron acquisition pathway from enterohemorrhagic e. coli. [Thesis]. University of Georgia; 2017. Available from: http://hdl.handle.net/10724/36798

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Vanderbilt University

20. Spencer, Cierra Tamese. Biochemical characterization of a Pseudomonas aeruginosa phospholipase D.

Degree: PhD, Pharmacology, 2015, Vanderbilt University

URL: http://hdl.handle.net/1803/10428

► Phospholipase D (PLD) is a ubiquitous enzyme found in prokaryotic and eukaryotic organisms that generates phosphatidic acid. A number of human bacterial pathogens produce PLD… (more)

Subjects/Keywords: phospholipase D; enzymology; bacterial virulence

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APA (6^th Edition):

Spencer, C. T. (2015). Biochemical characterization of a Pseudomonas aeruginosa phospholipase D. (Doctoral Dissertation). Vanderbilt University. Retrieved from http://hdl.handle.net/1803/10428

Chicago Manual of Style (16^th Edition):

Spencer, Cierra Tamese. “Biochemical characterization of a Pseudomonas aeruginosa phospholipase D.” 2015. Doctoral Dissertation, Vanderbilt University. Accessed January 20, 2021. http://hdl.handle.net/1803/10428.

MLA Handbook (7^th Edition):

Spencer, Cierra Tamese. “Biochemical characterization of a Pseudomonas aeruginosa phospholipase D.” 2015. Web. 20 Jan 2021.

Vancouver:

Spencer CT. Biochemical characterization of a Pseudomonas aeruginosa phospholipase D. [Internet] [Doctoral dissertation]. Vanderbilt University; 2015. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1803/10428.

Council of Science Editors:

Spencer CT. Biochemical characterization of a Pseudomonas aeruginosa phospholipase D. [Doctoral Dissertation]. Vanderbilt University; 2015. Available from: http://hdl.handle.net/1803/10428

21. Ochem, Alexander. Properties of two DNA helicases of human cells.

Degree: PhD, 1999, Open University

URL: http://oro.open.ac.uk/65337/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299015

► DNA helicases are ubiquitous, non-specific dsDNA unwinding enzymes involved in all aspects of DNA metabolism. In the present work, I describe the properties of two… (more)

Subjects/Keywords: 572; Enzymology

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APA (6^th Edition):

Ochem, A. (1999). Properties of two DNA helicases of human cells. (Doctoral Dissertation). Open University. Retrieved from http://oro.open.ac.uk/65337/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299015

Chicago Manual of Style (16^th Edition):

Ochem, Alexander. “Properties of two DNA helicases of human cells.” 1999. Doctoral Dissertation, Open University. Accessed January 20, 2021. http://oro.open.ac.uk/65337/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299015.

MLA Handbook (7^th Edition):

Ochem, Alexander. “Properties of two DNA helicases of human cells.” 1999. Web. 20 Jan 2021.

Vancouver:

Ochem A. Properties of two DNA helicases of human cells. [Internet] [Doctoral dissertation]. Open University; 1999. [cited 2021 Jan 20]. Available from: http://oro.open.ac.uk/65337/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299015.

Council of Science Editors:

Ochem A. Properties of two DNA helicases of human cells. [Doctoral Dissertation]. Open University; 1999. Available from: http://oro.open.ac.uk/65337/ ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299015

University of British Columbia

22. Horsman, Geoffrey. Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls.

Degree: PhD, Biochemistry and Molecular Biology, 2008, University of British Columbia

URL: http://hdl.handle.net/2429/295

► Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway… (more)

▼ Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme.

Subjects/Keywords: enzymology; biodegradation

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APA (6^th Edition):

Horsman, G. (2008). Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls. (Doctoral Dissertation). University of British Columbia. Retrieved from http://hdl.handle.net/2429/295

Chicago Manual of Style (16^th Edition):

Horsman, Geoffrey. “Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls.” 2008. Doctoral Dissertation, University of British Columbia. Accessed January 20, 2021. http://hdl.handle.net/2429/295.

MLA Handbook (7^th Edition):

Horsman, Geoffrey. “Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls.” 2008. Web. 20 Jan 2021.

Vancouver:

Horsman G. Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls. [Internet] [Doctoral dissertation]. University of British Columbia; 2008. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2429/295.

Council of Science Editors:

Horsman G. Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls. [Doctoral Dissertation]. University of British Columbia; 2008. Available from: http://hdl.handle.net/2429/295

University of Michigan

23. Kaitany, Kipchumba. Substrate Recognition Mechanism of Protein-only RNase P.

Degree: PhD, Biological Chemistry, 2020, University of Michigan

URL: http://hdl.handle.net/2027.42/163075

► Ribonuclease P (RNase P) is the enzyme responsible for catalyzing the removal of the 5’ leader sequence from precursor transfer RNA (pre-tRNA) during the essential… (more)

▼ Ribonuclease P (RNase P) is the enzyme responsible for catalyzing the removal of the 5’ leader sequence from precursor transfer RNA (pre-tRNA) during the essential maturation process of transfer RNA (tRNA). While RNase P was first discovered in lower order organisms as an RNA-based ribozyme, recent work has revealed the existence of a protein-only RNase P (PRORP) within Eukaryotes. The existence of these two types of independently evolved RNase P enzymes, provides the rare opportunity to compare convergent evolutionary strategies of RNA- and protein-based catalysts. Previous work revealed that both the proteinaceous and RNA-based enzymes achieve nucleolytic activity through the same general two-metal ion mechanism. However, how PRORP recognizes tRNA substrates and whether this mechanism is similar to RNA-based RNase P is poorly understood. This work investigates the substrate recognition strategy of PRORP. Mutagenesis and in vitro binding and catalytic activity assays identified several residues within the conserved pentatricopeptide repeat (PPR) domain of Arabidopsis PRORP1 which form binding interactions with pre-tRNA. A crystal structure of the PPR domain bound to tRNA is solved. Residues critical for binding are shown to form interactions with conserved regions of tRNA. This mode of RNA recognition by a PPR domain is novel, differing from the established sequence-specific RNA model. Interestingly, RNA-based RNase P uses a similar mechanism to detect tRNAs and this is additional evidence of convergent evolution between RNA and protein-only forms of RNase P. Overall this work identifies the mechanism of PPR domain pre-tRNA recognition, providing a foundation for the elucidating the function and mechanism of other PPR-motif containing proteins. B Advisors/Committee Members: Fierke, Carol A (committee member), Koutmos, Markos (committee member), Frank, Aaron Terrence (committee member), Freddolino, Peter Louis (committee member), O'Brien, Patrick (committee member), Palfey, Bruce Allan (committee member).

Subjects/Keywords: Biochemistry; Enzymology; Biological Chemistry; Science

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APA (6^th Edition):

Kaitany, K. (2020). Substrate Recognition Mechanism of Protein-only RNase P. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/163075

Chicago Manual of Style (16^th Edition):

Kaitany, Kipchumba. “Substrate Recognition Mechanism of Protein-only RNase P.” 2020. Doctoral Dissertation, University of Michigan. Accessed January 20, 2021. http://hdl.handle.net/2027.42/163075.

MLA Handbook (7^th Edition):

Kaitany, Kipchumba. “Substrate Recognition Mechanism of Protein-only RNase P.” 2020. Web. 20 Jan 2021.

Vancouver:

Kaitany K. Substrate Recognition Mechanism of Protein-only RNase P. [Internet] [Doctoral dissertation]. University of Michigan; 2020. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2027.42/163075.

Council of Science Editors:

Kaitany K. Substrate Recognition Mechanism of Protein-only RNase P. [Doctoral Dissertation]. University of Michigan; 2020. Available from: http://hdl.handle.net/2027.42/163075

University of Texas – Austin

24. Szu, Ping-Hui, 1978-. The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme.

Degree: PhD, Chemistry, 2008, University of Texas – Austin

URL: http://hdl.handle.net/2152/3956

► D-Desosamine, a 3-(dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae, plays an essential role in… (more)

▼ D-Desosamine, a 3-(dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae, plays an essential role in conferring biological activities to its parent aglycones. The proteins encoded by the desI and desII genes in the methymycin/pikromycin biosynthetic gene cluster have been proposed to catalyze C-4 deoxygenation in D-desosamine biosynthesis. DesI is a pyridoxal 5'-phosphate-dependent C4-aminotransferase and catalyzes a transamination reaction converting thymidine diphosphate (TDP)-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-glucose. DesII, which contains a [4Fe-4S] cluster binding motif, CXXXCXXC, has been identified as a member of the radical S-adenosylmethionine (SAM) enzyme superfamily by sequence analysis. To study the catalytic function of DesII, the desII gene was heterologously overexpressed in Escherichia coli and the DesII protein was purified to near homogeneity. Biochemical studies clearly established that the substrate for DesII is TDP-4-amino-4,6-dideoxy-D-glucose, and DesI and DesII function independently to carry out C-4 deoxygenation. DesII requires a [4Fe-4S]¹⁺ center and Sadenosylmethionine for activity. Accordingly, the originally proposed mechanism for C-4 deoxygenation in which DesI and DesII function together was revised. Two possible mechanisms have subsequently been proposed for the DesII reaction. The DesII catalysis is likely initiated by the formation of a 5'-deoxyadenosyl radical followed by the C-3 hydrogen atom abstraction. In the first possible route, the key step is a radical-induced deamination followed by the readdition of ammonia to the resulting cation radical intermediate, which is effectively a 1,2-amino shift, to form an aminol radical. Alternatively, the reaction may involve deprotonation of the 3-hydroxyl group to yield a ketyl radical anion to facilitate the [beta]-elimination of the ammonia group. Interestingly, DesII is flexible towards TDP-D-quinovose and TDP-3-amino-3,6-dideoxy-D-glucose. A possible biological reducing system, flavodoxin, flavodoxin reductase, and NADPH, for the reduction of the [4Fe-4S]²⁺ cluster, was also identified. Deuterium incorporation into SAM using C-3 deuterium-labeled substrate provides solid evidence for C-3 hydrogen atom abstraction by the 5'-deoxyadenosyl radical in the proposed mechanism. TDP-3-fluoro-3,6-dideoxy-D-glucose serves as a competitive inhibitor for DesII, which is in favor of deprotonation of the C-3 hydroxyl group being involved in DesII catalysis. Advisors/Committee Members: Liu, Hung-wen, 1952- (advisor).

Subjects/Keywords: Biosynthesis; Enzymology

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APA (6^th Edition):

Szu, Ping-Hui, 1. (2008). The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme. (Doctoral Dissertation). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/3956

Chicago Manual of Style (16^th Edition):

Szu, Ping-Hui, 1978-. “The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme.” 2008. Doctoral Dissertation, University of Texas – Austin. Accessed January 20, 2021. http://hdl.handle.net/2152/3956.

MLA Handbook (7^th Edition):

Szu, Ping-Hui, 1978-. “The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme.” 2008. Web. 20 Jan 2021.

Vancouver:

Szu, Ping-Hui 1. The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme. [Internet] [Doctoral dissertation]. University of Texas – Austin; 2008. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2152/3956.

Council of Science Editors:

Szu, Ping-Hui 1. The biosynthesis of TDP-D-Desosamine: characterization and mechanistic studies of DesII, a radical S-adenosylmethionine-dependent enzyme. [Doctoral Dissertation]. University of Texas – Austin; 2008. Available from: http://hdl.handle.net/2152/3956

University of Utah

25. Plager, John Everett. Enzymic reactions of the adrenal gland involved in the hydroxylation of carbons 17 and 21 of the pregnene nucleus.

Degree: PhD, Biochemistry;, 1953, University of Utah

URL: http://content.lib.utah.edu/cdm/singleitem/collection/etd2/id/1012/rec/425

► Two enzyme systems have been demonstrated in beef adrenal homogenates. These systems will introduce the 17 alpha-hydroxyl group into the progesterone molecule and oxidize the… (more)

Subjects/Keywords: Enzymology; Steroids Synthesis

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APA (6^th Edition):

Plager, J. E. (1953). Enzymic reactions of the adrenal gland involved in the hydroxylation of carbons 17 and 21 of the pregnene nucleus. (Doctoral Dissertation). University of Utah. Retrieved from http://content.lib.utah.edu/cdm/singleitem/collection/etd2/id/1012/rec/425

Chicago Manual of Style (16^th Edition):

Plager, John Everett. “Enzymic reactions of the adrenal gland involved in the hydroxylation of carbons 17 and 21 of the pregnene nucleus.” 1953. Doctoral Dissertation, University of Utah. Accessed January 20, 2021. http://content.lib.utah.edu/cdm/singleitem/collection/etd2/id/1012/rec/425.

MLA Handbook (7^th Edition):

Plager, John Everett. “Enzymic reactions of the adrenal gland involved in the hydroxylation of carbons 17 and 21 of the pregnene nucleus.” 1953. Web. 20 Jan 2021.

Vancouver:

Plager JE. Enzymic reactions of the adrenal gland involved in the hydroxylation of carbons 17 and 21 of the pregnene nucleus. [Internet] [Doctoral dissertation]. University of Utah; 1953. [cited 2021 Jan 20]. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd2/id/1012/rec/425.

Council of Science Editors:

Plager JE. Enzymic reactions of the adrenal gland involved in the hydroxylation of carbons 17 and 21 of the pregnene nucleus. [Doctoral Dissertation]. University of Utah; 1953. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd2/id/1012/rec/425

26. Santos, Tamara Angelo de Oliveira. Peptidases e lipases produzidas pelo fungo Fusarium oxysporum: caracterização e microencapsulação por spray drying.

Degree: Mestrado, Medicamentos e Cosméticos, 2012, University of São Paulo

URL: http://www.teses.usp.br/teses/disponiveis/60/60137/tde-28062012-161319/ ;

►

Duas variações de resíduo agroindustrial foram analisadas como meio de cultura para o bioprocesso de fermentação semissólida pelo fungo Fusarium oxysporum, com o objetivo de… (more)

▼

Duas variações de resíduo agroindustrial foram analisadas como meio de cultura para o bioprocesso de fermentação semissólida pelo fungo Fusarium oxysporum, com o objetivo de obter a melhor produção de peptidases e lipases. Essas enzimas foram microencapsuladas por spray drying, visando garantir sua estabilidade e investigar outros prováveis benefícios obtidos pela técnica. A utilização de planejamento experimental permitiu analisar os efeitos e interações entre as variáveis operacionais do processo (temperatura de secagem, proporção de adjuvantes e relação entre adjuvantes). A caracterização bioquímica e físico-química do extrato enzimático e das micropartículas também foram estudadas. O emprego de farelo de trigo como substrato demonstrou maior produção enzimática que o uso de farelo de algodão. A fermentação produziu uma serinopeptidase e uma lipase, ambas com característica alcalina, com alta estabilidade em ampla faixa de pH e certa estabilidade em diferentes temperaturas. Para ambas as enzimas, observou-se modulação positiva da atividade frente à maioria dos íons estudados e forte inibição pelo surfactante SDS, enquanto a lipase demonstrou superatividade frente a CTAB. A caracterização enzimática permite sugerir a aplicação dessas enzimas na formulação de detergentes enzimáticos, indústria de couro, indústria de papel, agroquímicos, síntese de biopolímeros e biodísel. No processo de microencapsulação, a temperatura foi a variável operacional mais importante para a estabilidade, enquanto a quantidade de adjuvantes em relação à quantidade de extrato enzimático influenciou nas condições de manipulação. O estudo demonstrou que a técnica de microencapsulação por spray drying resultou em grande benefício no armazenamento das enzimas, por aumentar consideravelmente sua estabilidade e melhorar as propriedades físicas do extrato.

Two variations of agroindustrial residue were analyzed as culture medium for the bioprocess of solid-state fermentation by the fungus Fusarium oxysporum, in order to achieve the best production of peptidases and lipases. These enzymes were microencapsulated by spray drying in order to ensure its stability and investigate other potential benefits obtained by the technique. The use of experimental design allowed us to analyze the effects and interactions between the operating variables of the process (drying temperature, proportion of adjuvants and relation among adjuvants). Biochemical and physico-chemical characterization of the enzymatic extract and of the microparticles were also studied. The use of wheat bran as substrate demonstrated a greater enzyme production then the use of cottonseed meal. The fermentation produced serinepeptidases and lipases, both alkaline, with high stability over a wide pH range and some stability at different temperatures. For both enzymes, there was up regulation of activity with most of the ions analyzed and strong inhibition against the surfactant SDS, whereas lipase demonstrated superactivity against CTAB. The enzymatic characterization suggests the…

Advisors/Committee Members: Cabral, Hamilton.

Subjects/Keywords: bioencapsulação; bioencapsulation; biotechnology; biotecnologia; enzimologia; enzymology

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APA (6^th Edition):

Santos, T. A. d. O. (2012). Peptidases e lipases produzidas pelo fungo Fusarium oxysporum: caracterização e microencapsulação por spray drying. (Masters Thesis). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/60/60137/tde-28062012-161319/ ;

Chicago Manual of Style (16^th Edition):

Santos, Tamara Angelo de Oliveira. “Peptidases e lipases produzidas pelo fungo Fusarium oxysporum: caracterização e microencapsulação por spray drying.” 2012. Masters Thesis, University of São Paulo. Accessed January 20, 2021. http://www.teses.usp.br/teses/disponiveis/60/60137/tde-28062012-161319/ ;.

MLA Handbook (7^th Edition):

Santos, Tamara Angelo de Oliveira. “Peptidases e lipases produzidas pelo fungo Fusarium oxysporum: caracterização e microencapsulação por spray drying.” 2012. Web. 20 Jan 2021.

Vancouver:

Santos TAdO. Peptidases e lipases produzidas pelo fungo Fusarium oxysporum: caracterização e microencapsulação por spray drying. [Internet] [Masters thesis]. University of São Paulo; 2012. [cited 2021 Jan 20]. Available from: http://www.teses.usp.br/teses/disponiveis/60/60137/tde-28062012-161319/ ;.

Council of Science Editors:

Santos TAdO. Peptidases e lipases produzidas pelo fungo Fusarium oxysporum: caracterização e microencapsulação por spray drying. [Masters Thesis]. University of São Paulo; 2012. Available from: http://www.teses.usp.br/teses/disponiveis/60/60137/tde-28062012-161319/ ;

27. Stull, Frederick W. Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase.

Degree: PhD, Chemical Biology, 2014, University of Michigan

URL: http://hdl.handle.net/2027.42/110420

► Pyrimidines are essential components of nucleic acids. In biology, they undergo a number of redox reactions catalyzed by flavin-dependent enzymes. This thesis investigates the mechanism… (more)

▼ Pyrimidines are essential components of nucleic acids. In biology, they undergo a number of redox reactions catalyzed by flavin-dependent enzymes. This thesis investigates the mechanism of substrate recognition and activation of two flavin-dependent enzymes with pyrimidine-containing substrates: flavin-dependent thymidylate synthase (FDTS) and tRNA-dihydrouridine synthase (DUS). FDTS catalyzes the reductive methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP) into thymine, whereas DUS reduces specific uracils in tRNA to dihydrouracil. NMR data indicate that FDTS ionizes N3 of the uracil in dUMP using an active-site arginine, which is proposed to initiate catalysis by enhancing the nucleophilicity of C5 of the uracil. Biochemical data on dUMP analogs suggests that the phosphate of dUMP acts as the base that removes the proton from C5 of dUMP during the FDTS-catalyzed reaction. Notably, both ionization of N3 and acid-base catalysis by the phosphate of dUMP are not implicated in the mechanism used by human thymidylate synthase. Several equilibrium and kinetic methods were used to study the mechanism of deoxynucleotide recognition by Thermotoga maritima FDTS. FDTS binds deoxynucleotides with ~200-fold weaker affinity when the flavin is reduced relative to when it is oxidized, and the differences in affinity are largely due to differences in the dissociation rate constant. There is also a temperature-dependent effect on the mechanism by which FDTS binds deoxynucleotides – below 45°C the FDTS homotetramer behaves as a dimer-of-dimers with deoxynucleotide binding while at temperatures above 45°C the four subunits of FDTS bind deoxynucleotides identically. tRNAs contain a number of nucleobase and ribose modifications – including dihydrouracil – at different positions of the tRNA. Previous work has shown that yeast tRNALeu-CAA reacts poorly with yeast DUS2 unless it contains other modifications, suggesting that tRNA modifications are ordered. The reactivity of yeast DUS2 with other unmodified tRNAs was investigated; unmodified yeast tRNAAsp, tRNAAla, and tRNAHis2 all reacted rapidly with yeast DUS2, indicating that not all tRNAs require prior modifications to react rapidly with yeast DUS2. Native gel electrophoresis showed that unmodified tRNALeu-CAA misfolds, explaining its poor reactivity with yeast DUS2. Advisors/Committee Members: Palfey, Bruce A. (committee member), Zuiderweg, Erik R P (committee member), O'Brien, Patrick (committee member), Garcia, George A. (committee member).

Subjects/Keywords: Mechanistic enzymology; Flavin; Pyrimidine; Biological Chemistry; Science

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APA (6^th Edition):

Stull, F. W. (2014). Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/110420

Chicago Manual of Style (16^th Edition):

Stull, Frederick W. “Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase.” 2014. Doctoral Dissertation, University of Michigan. Accessed January 20, 2021. http://hdl.handle.net/2027.42/110420.

MLA Handbook (7^th Edition):

Stull, Frederick W. “Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase.” 2014. Web. 20 Jan 2021.

Vancouver:

Stull FW. Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase. [Internet] [Doctoral dissertation]. University of Michigan; 2014. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2027.42/110420.

Council of Science Editors:

Stull FW. Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism: Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase. [Doctoral Dissertation]. University of Michigan; 2014. Available from: http://hdl.handle.net/2027.42/110420

Vanderbilt University

28. Albertolle, Matthew Edward. SULFENYLATION OF CYTOCHROMES P450 IN RESPONSE TO REDOX ALTERATION.

Degree: PhD, Biochemistry, 2019, Vanderbilt University

URL: http://hdl.handle.net/1803/10450

► Mammalian cytochrome P450 (P450) enzymes catalyze complex reactions involved in the biosynthesis of endogenous metabolites such as steroids, vitamins, and hormones. Additionally, several enzymes in… (more)

Subjects/Keywords: Cytochrome P450; Redox; Enzymology; Proteomics; Sulfenic Acid

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APA (6^th Edition):

Albertolle, M. E. (2019). SULFENYLATION OF CYTOCHROMES P450 IN RESPONSE TO REDOX ALTERATION. (Doctoral Dissertation). Vanderbilt University. Retrieved from http://hdl.handle.net/1803/10450

Chicago Manual of Style (16^th Edition):

Albertolle, Matthew Edward. “SULFENYLATION OF CYTOCHROMES P450 IN RESPONSE TO REDOX ALTERATION.” 2019. Doctoral Dissertation, Vanderbilt University. Accessed January 20, 2021. http://hdl.handle.net/1803/10450.

MLA Handbook (7^th Edition):

Albertolle, Matthew Edward. “SULFENYLATION OF CYTOCHROMES P450 IN RESPONSE TO REDOX ALTERATION.” 2019. Web. 20 Jan 2021.

Vancouver:

Albertolle ME. SULFENYLATION OF CYTOCHROMES P450 IN RESPONSE TO REDOX ALTERATION. [Internet] [Doctoral dissertation]. Vanderbilt University; 2019. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1803/10450.

Council of Science Editors:

Albertolle ME. SULFENYLATION OF CYTOCHROMES P450 IN RESPONSE TO REDOX ALTERATION. [Doctoral Dissertation]. Vanderbilt University; 2019. Available from: http://hdl.handle.net/1803/10450

Texas A&M University

29. Liu, Zhen. Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis.

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

URL: http://hdl.handle.net/1969.1/151650

► Mycobacterium tuberculosis (M. tuberculosis) contains a wide array of genes responsible for the synthesis and secretion of a variety of bioactive lipids. The genes represent… (more)

▼ Mycobacterium tuberculosis (M. tuberculosis) contains a wide array of genes responsible for the synthesis and secretion of a variety of bioactive lipids. The genes represent attractive drug-targets due to their involvement in essential cell cycles, the implication in pathogenesis, and the interference with therapeutics. In this thesis, I report our efforts to understand the biological functions of, and to develop inhibitors against, multiple genes related to M. tuberculosis lipid metabolism. Firstly, dioctylamine, a substrate mimic of the mycolic acid cyclopropane synthases, is shown to inhibit CmaA2 in vitro. Its inhibition action is explained by the structural characterization. Together with our collaborators, we have found dioctylamine able to intervene multiple mycolic acid cyclopropane synthases in vivo, and hence established the first model study for the single-drug-multiple-target strategy to inhibit the mycolic acid biosynthesis of M. tuberculosis. In addition, dioctylamine can serve as the platform for the design of more potent and selective drugs in the future. Secondly, the action mechanism of isoniazid and ethionamide, both of which are pro-drugs targeting the mycolic acid biosynthesis, is explored via biochemical, X-ray crystallographic or modeling studies. We have determined that the intracellular target of isoniazid is the enoyl reductase InhA; and we have discovered the correlation between mycothiol and ethionamide susceptibility. Thirdly, I have investigated the function and mechanism of FadD10, an enzyme involved in the synthesis of a virulence-related lipopeptide. The results reveal that FadD10 was mis-annotated as a fatty acyl-CoA ligase, but it indeed transfers fatty acids to an acyl carrier protein (Rv0100). Further crystallographic characterization provides the molecular basis for the mechanism of FadD10, leading to the discovery of a new type of adenylate-forming enzyme. Advisors/Committee Members: Sacchettini, James C. (advisor), Barondeau, David (committee member), Burgess, Kevin (committee member), Igumenova, Tatyana (committee member).

Subjects/Keywords: Structural biology; Enzymology; Drug discovery; Lipid metabolism

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APA (6^th Edition):

Liu, Z. (2013). Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/151650

Chicago Manual of Style (16^th Edition):

Liu, Zhen. “Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis.” 2013. Doctoral Dissertation, Texas A&M University. Accessed January 20, 2021. http://hdl.handle.net/1969.1/151650.

MLA Handbook (7^th Edition):

Liu, Zhen. “Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis.” 2013. Web. 20 Jan 2021.

Vancouver:

Liu Z. Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis. [Internet] [Doctoral dissertation]. Texas A&M University; 2013. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1969.1/151650.

Council of Science Editors:

Liu Z. Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis. [Doctoral Dissertation]. Texas A&M University; 2013. Available from: http://hdl.handle.net/1969.1/151650

Texas A&M University

30. Kim, Jungwook. Molecular engineering of oligomerization and metabolite channeling through a molecular tunnel of carbamoyl phosphate synthetase.

Degree: PhD, Chemistry, 2004, Texas A&M University

URL: http://hdl.handle.net/1969.1/332

► The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the… (more)

▼ The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (αβ)4 tetramer through disruption of the molecular binding interactions between monomeric units. The results are consistent with a model for the structure of the (αβ)2 dimer that is formed through molecular contact between two pairs of allosteric domains. No significant dependence of the specific catalytic activity on the protein concentration could be detected. The molecular tunnel within CPS was inspected in order to characterize the role on kinetic properties. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The kinetic data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain to yield a final product carbamoyl phosphate. In addition, a unique feature of the carbamate tunnel has been noted where five highly conserved glutamates are located on a particular interior face of the tunnel. It has been postulated that the negative charge stabilizes the acid-labile intermediate, and facilitates catalysis. Also, the proposed gate keeping residues, Arg-306 and Arg-848, have been mutated to alanines to test their roles. However, since the arginines directly interact with MgATP, the mutation appeared to interrupt the binding of the substrate. The ammonia tunnel has been engineered to contain a hole to further support the proposed role of the tunnel that it is utilized in guiding diffusion of ammonia from the site of glutamine hydrolysis to the subsequent active site in the large subunit. Triple mutant αP360A/αH361A/βR265A exhibited kinetic behaviors consistent with a model of an impaired channeling. Advisors/Committee Members: Raushel, Frank M. (advisor), Lindahl, Paul A. (committee member), DeRose, Victoria J. (committee member), Reinhart, Gregory D. (committee member).

Subjects/Keywords: biochemistry; enzymology; CPS

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

APA (6^th Edition):

Kim, J. (2004). Molecular engineering of oligomerization and metabolite channeling through a molecular tunnel of carbamoyl phosphate synthetase. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/332

Chicago Manual of Style (16^th Edition):

Kim, Jungwook. “Molecular engineering of oligomerization and metabolite channeling through a molecular tunnel of carbamoyl phosphate synthetase.” 2004. Doctoral Dissertation, Texas A&M University. Accessed January 20, 2021. http://hdl.handle.net/1969.1/332.

MLA Handbook (7^th Edition):

Kim, Jungwook. “Molecular engineering of oligomerization and metabolite channeling through a molecular tunnel of carbamoyl phosphate synthetase.” 2004. Web. 20 Jan 2021.

Vancouver:

Kim J. Molecular engineering of oligomerization and metabolite channeling through a molecular tunnel of carbamoyl phosphate synthetase. [Internet] [Doctoral dissertation]. Texas A&M University; 2004. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1969.1/332.

Council of Science Editors:

Kim J. Molecular engineering of oligomerization and metabolite channeling through a molecular tunnel of carbamoyl phosphate synthetase. [Doctoral Dissertation]. Texas A&M University; 2004. Available from: http://hdl.handle.net/1969.1/332

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