2026-09-20T10:42:25Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/1096812026-06-16T18:14:57Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
JoAnne Stubbe.
Parker, Mackenzie James
Massachusetts Institute of Technology. Department of Chemistry.
Massachusetts Institute of Technology. Department of Chemistry
2017-06-06T19:25:08Z
2017-06-06T19:25:08Z
2017
2017
http://hdl.handle.net/1721.1/109681
988746188
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.
Page 490 blank. Cataloged from PDF version of thesis.
Includes bibliographical references.
Ribonucleotide reductases (RNRs) catalyze the reduction of nucleotides to 2'-deoxynucleotides in all organisms. Class lb RNRs consist of two subunits: a houses the catalytic and allosteric effector binding sites, and p houses a catalytically essential dimanganic-tyrosyl radical (Mn(III)2-Y*). The allosteric regulation of lb RNR activity has only been studied with the Salmonella enterica enzyme, which exhibits substrate specificity allosteric regulation by ATP and 2'-deoxynucleoside 5'-triphosphates (dNTPs), but not overall activity regulation by ATP and dATP. However, the S. enterica enzyme is not a good general model for Ib RNRs because it is not essential under most growth conditions, including pathogenesis. Other bacteria pathogenic to humans utilize lb RNRs as their sole source of dNTPs for DNA replication and repair. As RNR regulation plays a critical role in the high fidelity of these processes, the allosteric regulation of lb RNRs used as the primary dNTP supplier for a bacterium should be distinct from the S. enterica enzyme and, therefore, could provide a potential target for therapeutic development. Herein, the results of characterizing the allosteric regulation of the Ib RNR from the model organism Bacillus subtilis are presented. To facilitate these studies, we identified, cloned, and isolated the physiological reductant for RNR (thioredoxin/thioredoxin reductase/NADPH), thus allowing us to monitor activity spectrophotometrically. We discovered the effector dATP was a potent inhibitor of enzymatic activity at physiologically relevant concentrations, thereby demonstrating the first example of overall activity allosteric regulation in a class lb system. In other RNRs, overall activity regulation is mediated by a domain called the ATP-cone. This domain is absent from the B. subtilis enzyme; therefore, the inhibition represents a new mechanism of overall activity regulation. Analytical ultracentrifugation studies suggest dATP inhibition may be mediated by formation of large protein complexes. Biophysical studies also led to the discovery of tightly bound dAMP associated with a that increases the susceptibility of RNR to dATP inhibition. The potential physiological importance of dAMP is supported by studies examining YmaB, the unique fourth member of the B. subtilis RNR operon, which revealed this enzyme can hydrolyze dATP into dAMP and pyrophosphate and, therefore, might insert dAMP into a.
by Mackenzie James Parker.
Ph.D.
490 pages
eng
Massachusetts Institute of Technology
MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.
http://dspace.mit.edu/handle/1721.1/7582
Chemistry.
Discovery and investigation of the novel overall activity allosteric regulation of the Bacillus subtilis class Ib ribonucleotide reductase
Thesis
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<Abstract>Ribonucleotide reductases (RNRs) catalyze the reduction of nucleotides to 2'-deoxynucleotides in all organisms. Class lb RNRs consist of two subunits: a houses the catalytic and allosteric effector binding sites, and p houses a catalytically essential dimanganic-tyrosyl radical (Mn(III)2-Y*). The allosteric regulation of lb RNR activity has only been studied with the Salmonella enterica enzyme, which exhibits substrate specificity allosteric regulation by ATP and 2'-deoxynucleoside 5'-triphosphates (dNTPs), but not overall activity regulation by ATP and dATP. However, the S. enterica enzyme is not a good general model for Ib RNRs because it is not essential under most growth conditions, including pathogenesis. Other bacteria pathogenic to humans utilize lb RNRs as their sole source of dNTPs for DNA replication and repair. As RNR regulation plays a critical role in the high fidelity of these processes, the allosteric regulation of lb RNRs used as the primary dNTP supplier for a bacterium should be distinct from the S. enterica enzyme and, therefore, could provide a potential target for therapeutic development. Herein, the results of characterizing the allosteric regulation of the Ib RNR from the model organism Bacillus subtilis are presented. To facilitate these studies, we identified, cloned, and isolated the physiological reductant for RNR (thioredoxin/thioredoxin reductase/NADPH), thus allowing us to monitor activity spectrophotometrically. We discovered the effector dATP was a potent inhibitor of enzymatic activity at physiologically relevant concentrations, thereby demonstrating the first example of overall activity allosteric regulation in a class lb system. In other RNRs, overall activity regulation is mediated by a domain called the ATP-cone. This domain is absent from the B. subtilis enzyme; therefore, the inhibition represents a new mechanism of overall activity regulation. Analytical ultracentrifugation studies suggest dATP inhibition may be mediated by formation of large protein complexes. Biophysical studies also led to the discovery of tightly bound dAMP associated with a that increases the susceptibility of RNR to dATP inhibition. The potential physiological importance of dAMP is supported by studies examining YmaB, the unique fourth member of the B. subtilis RNR operon, which revealed this enzyme can hydrolyze dATP into dAMP and pyrophosphate and, therefore, might insert dAMP into a.</Abstract>
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