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Title: Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Authors:
; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1398019
Grant/Contract Number:
AC02-06CH11357
Resource Type:
Journal Article: Publisher's Accepted Manuscript
Journal Name:
Bioorganic and Medicinal Chemistry
Additional Journal Information:
Journal Volume: 24; Journal Issue: 19; Related Information: CHORUS Timestamp: 2017-10-05 03:15:19; Journal ID: ISSN 0968-0896
Publisher:
Elsevier
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, and Johnson, Michael E.. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase). United Kingdom: N. p., 2016. Web. doi:10.1016/j.bmc.2016.07.055.
Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, & Johnson, Michael E.. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase). United Kingdom. doi:10.1016/j.bmc.2016.07.055.
Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, and Johnson, Michael E.. 2016. "Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)". United Kingdom. doi:10.1016/j.bmc.2016.07.055.
@article{osti_1398019,
title = {Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)},
author = {Rice, Amy J. and Lei, Hao and Santarsiero, Bernard D. and Lee, Hyun and Johnson, Michael E.},
abstractNote = {},
doi = {10.1016/j.bmc.2016.07.055},
journal = {Bioorganic and Medicinal Chemistry},
number = 19,
volume = 24,
place = {United Kingdom},
year = 2016,
month =
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.bmc.2016.07.055

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  • Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-89]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. Inmore » contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 {angstrom}, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.« less
  • Bacillus anthracis elaborates a poly-{gamma}-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the {gamma}-glutamyltranspeptidase CapD with and without {alpha}-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-{gamma}-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-{gamma}-glutamatemore » binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro{sup 427}, Gly{sup 428}, and Gly{sup 429} activate the catalytic residue of the enzyme, Thr{sup 352}, and stabilize an oxyanion hole via main chain amide hydrogen bonds.« less
  • Rhodanese homology domains (RHDs) play important roles in sulfur trafficking mechanisms essential to the biosynthesis of sulfur-containing cofactors and nucleosides. We have now determined the crystal structure at 2.10 {angstrom} resolution for the Bacillus anthracis coenzyme A-disulfide reductase isoform (BaCoADR-RHD) containing a C-terminal RHD domain; this is the first structural representative of the multidomain proteins class of the rhodanese superfamily. The catalytic Cys44 of the CoADR module is separated by 25 {angstrom} from the active-site Cys514' of the RHD domain from the complementary subunit. In stark contrast to the B. anthracis CoADR (Wallen, J. R., Paige, C., Mallett, T. C.,more » Karplus, P. A., and Claiborne, A. (2008) Biochemistry 47, 5182-5193), the BaCoADR-RHD isoform does not catalyze the reduction of coenzyme A-disulfide, although both enzymes conserve the Cys-SSCoA redox center. NADH titrations have been combined with a synchrotron reduction protocol for examination of the structural and redox behavior of the Cys44-SSCoA center. The synchrotron-reduced (Cys44 + CoASH) structure reveals ordered binding for the adenosine 3'-phosphate 5'-pyrophosphate moiety of CoASH, but the absence of density for the pantetheine arm indicates that it is flexible within the reduced active site. Steady-state kinetic analyses with the alternate disulfide substrates methyl methanethiolsulfonate (MMTS) and 5,5'-dithiobis(2-nitrobenzoate) (DTNB), including the appropriate Cys {yields} Ser mutants, demonstrate that MMTS reduction occurs within the CoADR active site. NADH-dependent DTNB reduction, on the other hand, requires communication between Cys44 and Cys514', and we propose that reduction of the Cys44-SSCoA disulfide promotes the transfer of reducing equivalents to the RHD, with the swinging pantetheine arm serving as a ca. 20 {angstrom} bridge.« less