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 glutathi one thiol/disulfide redox system employed by eukaryotes and many bacte ria. Instead, this organism produces CoA as its major low molecular we ight thiol. We report the identification and purification of the disul fide reductase component of this thiol/disulfide redox system. Coenzym e A disulfide reductase (CoADR) catalyzes the specific reduction of Co A disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of M-r 49,000 each. The visible absorbance spec trum is indicative of a flavoprotein with a lambda(max) = 452 nm. The liberated flavin from thermally denatured enzyme was identified as fla vin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a K-m for NADPH of 2 mu M and for CoA disulfide of 11 mu M. In additi on 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 emplo ys a single active site cysteine residue that forms a stable mixed dis ulfide with CoA during catalysis. These data suggest that S. aureus em ploys a thiol/disulfide redox system based on CoA/CoA-disulfide and Co ADR, an unorthodox new member of the pyridine nucleotide-disulfide red uctase superfamily.