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INVESTIGATION OF UNIQUE COENZYME A BIOSYNTHETIC AND REDOX FUNCTIONS IN Bacillus anthracis

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abstract
Coenzyme A (CoASH) replaces the common tripeptide thiol glutathione as the major low molecular-weight thiol in the human pathogen Bacillus anthracis. A novel type III pantothenate kinase (PanK) catalyzes the first committed step in the CoASH biosynthetic pathway in B. anthracis. The 2.0 Å crystal structure of the B. anthracis PanK (BaPanK) demonstrates that it is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily and modeling the pantothenate and ATP substrates into the active-site cleft provides a clear rationale for the absence of CoASH feedback inhibition. In addition, bioinformatics analyses indicate a widespread distribution of type III PanK isoforms. The gene encoding BaPanK, coaX, is transcribed as part of a tricistronic operon including hslO and cysK-1 loci, which encode the redox-regulated heat shock protein Hsp33 and cysteine synthase A, respectively. A conditional coaX mutant, in the absence of inducer, demonstrates exponential growth after a lag period of 8 h; this unanticipated result is due to a guanine to adenine suppressor mutation identified in the lac operator. Transcription of the tricistronic coaX-hslO-cysK-1 mRNA is observed in the suppressor mutant, as is observed with wild-type B. anthracis. Therefore, these data support BaPanK as an essential enzyme, thus contributing to its validation as a new antimicrobial target. The intracellular cellular thiol-disulfide redox status of B. anthracis is maintained by coenzyme A-disulfide reductase (BACoADR), which catalyzes the NAD(P)H-dependent reduction of coenzyme A-disulfide (CoAD) to 2 CoASH. B. anthracis also contains an additional CoADR isoform, coenzyme A-disulfide reductase-rhodanese homology domain (CoADR-RHD), which does not catalyze the reduction of CoAD and is implicated in to function in sulfur metabolism. In order to test the physiological contributions of BACoADR and CoADR-RHD in thiol-disulfide redox homeostasis, as well as germination and outgrowth of the B. anthracis endospore, in-frame deletion mutants of their respective encoding genes, cdr and cdrX, were constructed, singly and in combination. Both BACoADR and CoADR-RHD appear to have a role in the morphological transition from the endospore to the vegetative cell. However, CoADR-RHD appears to be the primary enzyme responsible for protection against diamide-induced disulfide stress. CoADRs represent unique members of the pyridine nucleotide disulfide oxidoreductase (PNDOR) family due to a single active-site cysteine, present as a stable Cys-SSCoA mixed disulfide. In order to distinguish CoADRs from the closely related NADH (per)oxidases and enable more accurate identification of CoADR proteins among its larger superfamily, hidden Markov model-based bioinformatics analyses have led to the development of CoADR-specific functional motifs.
subject
Bacillus anthracis
Coenzyme A
pantothenate kinase
coenzyme A disulfide reductase
contributor
Paige, Carleitta (author)
Sean D. Reid, PhD (committee chair)
Al Claiborne, PhD (committee member)
Thomas Hollis, PhD (committee member)
Dan Wozniak, PhD (committee member)
Derek Parsonage, PhD (committee member)
Jacquelyn Fetrow, PhD (committee member)
Nathan Fisher, PhD (committee member)
date
2009-03-19T14:17:45Z (accessioned)
2010-06-18T18:56:56Z (accessioned)
2009-03-19T14:17:45Z (available)
2010-06-18T18:56:56Z (available)
2009-03-19T14:17:45Z (issued)
degree
Biochemistry & Molecular Biology (discipline)
identifier
http://hdl.handle.net/10339/14656 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work immediately for access worldwide. (accessRights)
title
INVESTIGATION OF UNIQUE COENZYME A BIOSYNTHETIC AND REDOX FUNCTIONS IN Bacillus anthracis
type
Dissertation

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