Crystal Structure and Catalytic Properties of Bacillus anthracis CoADR-RHD: Implications for Flavin-Linked Sulfur Trafficking

Journal Article · Wed Dec 31 23:00:00 EST 2008 · Biochemistry

DOI:https://doi.org/10.1021/bi900887k· OSTI ID:980093

Wallen, J; Mallett, T; Boles, W; Parsonage, D; Furdui, C; Karplus, A; Claiborne, A

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., 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.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 980093
• Report Number(s): BNL--93011-2010-JA
• Journal Information: Biochemistry, Journal Name: Biochemistry Journal Issue: 40 Vol. 48
• Country of Publication: United States
• Language: English

Related Subjects

36 MATERIALS SCIENCE
43 PARTICLE ACCELERATORS
59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
ADENOSINE
ARMS
BACILLUS
BEHAVIOR
BIOCHEMISTRY
BIOSYNTHESIS
COENZYMES
COMMUNICATIONS
CRYSTAL STRUCTURE
DENSITY
DISULFIDES
ENZYMES
KINETICS
MUTANTS
NUCLEOSIDES
OXIDOREDUCTASES
PROTEINS
REDUCTION
RESOLUTION
SUBSTRATES
SULFUR
SYNCHROTRONS
YIELDS
national synchrotron light source