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You searched for +publisher:"University of Wisconsin – Milwaukee" +contributor:("Nicholas R. Silvaggi"). Showing records 1 – 2 of 2 total matches.

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University of Wisconsin – Milwaukee

1. Mueller, Lisa. Structural and Functional Characterization of Acetoacetate Decarboxylase-Like Enzymes.

Degree: PhD, Chemistry, 2016, University of Wisconsin – Milwaukee

The acetatoacetate decarboxylase-like superfamily (ADCSF) is a largely unexplored group of enzymes that may be a potential source of new biocatalysts. Bioinformatic analysis has grouped these approximately 2000 enzymes into seven different families based on comparison of predicted active site residues. To date, only the prototypical ADCs (Family I) that catalyze the decarboxylation of acetoacetate have been studied. Analysis of gene context suggests that Family V contains predominantly enzymes predicted to be involved in secondary metabolism. On average, these share about 20% sequence identity to the true ADCs. To learn more about the diversity of chemistries performed by members of Family V, we have been studying two enzymes annotated as "acetoacetate decarboxylase" in the GenBank database. These are Sbi_00515 from Streptomyces bingchenggensis and Swit_4259 from Sphingomonas wittichii. Steady state analyses of these enzymes demonstrate that both lack decarboxylase activity with any of the substrates tested. This was surprising given that the crystal structures of both enzymes show that their overall folds are almost indistinguishable from that of the prototypical ADCs, though the quaternary structures are different. An important observation from the bioinformatic and crystallographic analyses is that the catalytic lysine and putative acid/base catalyst residues of the true ADCs are retained in both groups of enzymes, but the active site architectures are different. Specifically, two residues shown to be important for the acetoacetate decarboxylase reaction, Arg29 and Glu61 in Clostridium acetobutylicum ADC (CaADC), are not retained in the Family V enzymes. Site-directed mutagenesis, steady state and transient kinetics, and mass spectroscopy data suggest evidence for reversible aldolase-dehydratase and retro-aldolase activities mediated through a Schiff-base mechanism. These are the first Schiff-base-forming aldolases that do not use the TIM barrel fold. Although the physiologically relevant reactions of these Family V enzymes are unknown, these studies illustrate that the ADC fold is a versatile platform that can be adapted to perform different chemistries. Advisors/Committee Members: Nicholas R. Silvaggi.

Subjects/Keywords: Biochemistry; Biophysics; Chemistry

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

APA (6th Edition):

Mueller, L. (2016). Structural and Functional Characterization of Acetoacetate Decarboxylase-Like Enzymes. (Doctoral Dissertation). University of Wisconsin – Milwaukee. Retrieved from https://dc.uwm.edu/etd/1296

Chicago Manual of Style (16th Edition):

Mueller, Lisa. “Structural and Functional Characterization of Acetoacetate Decarboxylase-Like Enzymes.” 2016. Doctoral Dissertation, University of Wisconsin – Milwaukee. Accessed August 20, 2019. https://dc.uwm.edu/etd/1296.

MLA Handbook (7th Edition):

Mueller, Lisa. “Structural and Functional Characterization of Acetoacetate Decarboxylase-Like Enzymes.” 2016. Web. 20 Aug 2019.

Vancouver:

Mueller L. Structural and Functional Characterization of Acetoacetate Decarboxylase-Like Enzymes. [Internet] [Doctoral dissertation]. University of Wisconsin – Milwaukee; 2016. [cited 2019 Aug 20]. Available from: https://dc.uwm.edu/etd/1296.

Council of Science Editors:

Mueller L. Structural and Functional Characterization of Acetoacetate Decarboxylase-Like Enzymes. [Doctoral Dissertation]. University of Wisconsin – Milwaukee; 2016. Available from: https://dc.uwm.edu/etd/1296


University of Wisconsin – Milwaukee

2. Han, Lanlan. Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis.

Degree: PhD, Chemistry, 2017, University of Wisconsin – Milwaukee

The first part of my thesis is focused on a new family of two-component response regulator proteins: Aspartate-Less Regulators (ALR). They lack the catalytic aspartate residue required for the phosphorylation mechanism of typical two component response regulators. We are using biophysical tools to characterize two proteins with redox-sensitive ALR domains: repressor of iron transport regulator (RitR) from Streptococcus pneumonia R6 and diguanylate cyclase Q15Z91 from Pseudoalteromonas atalantica. The structure of inactive RitRC128S monomer showed that the ALR domain and the DNA-binding domain are linked by an α-helix that runs the length of the entire protein, with C128 near the C-terminal end. Bioinformatic analysis of all streptococcal RitR homologs showed that Cys128 is strictly conserved, suggesting that RitR may be a novel redox sensor. Hydrogen peroxide was used to oxidize the cysteine thiol group to determine the structure of the oxidized, dimeric form of RitR. Oxidation of C128 to the disulfide caused a conformational change that caused the DBD to release from the ALR domain. Surprisingly, the freed DBD was observed bound to the ALR domain of the other, disulfide-linked molecule of RitR, recapitulating almost exactly the structure of the inactive, monomeric protein. An extended dimeric conformation was found in the RitRL86A/V93A variant. It binds to the target DNA according to gel filtration and differential scanning fluorimetry. The crystal structure of the RitRL86A/V93A ALR domain showed an unprecedented conformational change for a response regulator protein, where helix α4 is disordered and the two protomers swap their α5 helices to form the dimer. Combined with the C128D mutant in vivo studies, it seems that oxidation of C128 is part of the activation mechanism, but there must be an additional factor that leads to dimerization of the ALR domains. The second ALR protein Q15Z91 has R61 replacing the phosphorylatable aspartate residue in the ALR domain. According to the structure of Q15Z91 with GTP and c-di-GMP, purified Q15Z91 is an activated but product-inhibited dimer. C142 is conserved in the same position as C128 in RitR, and substitution demonstrated that C142 residue is also a redox sensor that involved in Q15Z91 activity regulation. The second part is a mechanistic enzymology project aimed at understanding the structure and mechanism of the novel pyridoxal-5’-phosphate (PLP)-dependent L-arginine hydroxylase/deaminase, MppP, from Streptomyces wadayamensis (SwMppP). SwMppP is predicted to be a type I/II aminotransferase based on primary sequence identity. However, NMR and ESI-MS results showed that SwMppP is not an aminotransferase, but rather a hydroxylase. The enzyme catalyzes the oxygen-dependent hydroxylation of L-arginine, forming 4-hydroxy-2-ketoarginine and the abortive side-product 2-ketoargine in a ratio of 1.7:1. This is exciting because SwMppP is the first PLP-dependent enzyme to react with oxygen in any context other than oxidative decarboxylation. The discovery of this new… Advisors/Committee Members: Nicholas R. Silvaggi.

Subjects/Keywords: Antibiotic Biosynthesis; Aspartate-Less Regulators; Enduracididine; Oxidase; Pyridoxal-5’-Phosphate; Redox Sensor; Biochemistry

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

APA (6th Edition):

Han, L. (2017). Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis. (Doctoral Dissertation). University of Wisconsin – Milwaukee. Retrieved from https://dc.uwm.edu/etd/1636

Chicago Manual of Style (16th Edition):

Han, Lanlan. “Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis.” 2017. Doctoral Dissertation, University of Wisconsin – Milwaukee. Accessed August 20, 2019. https://dc.uwm.edu/etd/1636.

MLA Handbook (7th Edition):

Han, Lanlan. “Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis.” 2017. Web. 20 Aug 2019.

Vancouver:

Han L. Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis. [Internet] [Doctoral dissertation]. University of Wisconsin – Milwaukee; 2017. [cited 2019 Aug 20]. Available from: https://dc.uwm.edu/etd/1636.

Council of Science Editors:

Han L. Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis. [Doctoral Dissertation]. University of Wisconsin – Milwaukee; 2017. Available from: https://dc.uwm.edu/etd/1636

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