Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme.
The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.
delCardayre
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Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. stephen_delcardayre@maxygen.com
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Newton
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Fahey
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Davies
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D001426:Bacterial Proteins / Q000302:isolation & purification; D001665:Binding Sites / Q000502:physiology; D003065:Coenzyme A / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005420:Flavoproteins / Q000737:chemistry; D019803:Glutathione Disulfide / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D015394:Molecular Structure; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013211:Staphylococcus aureus / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001426:Bacterial Proteins; D005420:Flavoproteins; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; C017145:coenzyme A disulfide; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C519574:coenzyme A disulfide reductase, Staphylococcus aureus; D003065:Coenzyme A; D019803:Glutathione Disulfide