title: Crystal structure and catalytic properties of Bacillus anthracis CoADR-RHD: implications for flavin-linked sulfur trafficking.
abstract: 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 A 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 A 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., 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 --> 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 A bridge.
authors.affiliation: Department of Internal Medicine, Wake Forest University Schoolof Medicine, Center for Structural Biology, Winston-Salem, North Carolina 27157, USA.
authors.forename: Jamie R
authors.initials: JR
authors.lastname: Wallen
authors.forename: T Conn
authors.initials: TC
authors.lastname: Mallett
authors.forename: William
authors.initials: W
authors.lastname: Boles
authors.forename: Derek
authors.initials: D
authors.lastname: Parsonage
authors.forename: Cristina M
authors.initials: CM
authors.lastname: Furdui
authors.forename: P Andrew
authors.initials: PA
authors.lastname: Karplus
authors.forename: Al
authors.initials: A
authors.lastname: Claiborne
mesh_terms: D001408:Bacillus anthracis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001693:Biological Transport, Active; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000378:metabolism; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000737:chemistry* / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D005415:Flavins; D007527:Isoenzymes; D013455:Sulfur; D009247:NADH, NADPH Oxidoreductases; C014703:disulfide reductase (NADH); D013884:Thiosulfate Sulfurtransferase; D003065:Coenzyme A