INVESTIGATION OF UNIQUE COENZYME A BIOSYNTHETIC AND REDOX FUNCTIONS IN Bacillus anthracis
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- title
- INVESTIGATION OF UNIQUE COENZYME A BIOSYNTHETIC AND REDOX FUNCTIONS IN Bacillus anthracis
- author
- Paige, Carleitta
- 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
- 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)
- type
- Dissertation