Purification and characterization of a Bacillus megaterium disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate.
An NADH-linked disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate moieties was purified 23,000-fold to homogeneity from spores of Bacillus megaterium. The enzyme had a native molecular weight of 122,000 with two apparently identical subunits, contained one molecule of flavin adenine dinucleotide per subunit, and was inhibited by the vicinal dithiol reagent arsenite. The enzyme was active only on disulfides containing pantethine 4',4"-diphosphate moieties, including pantethine 4',4"-diphosphate, oxidized coenzyme A, and coenzyme A in disulfide linkage to acyl carrier protein. However, the Km values for pantethine 4',4"-diphosphate and oxidized coenzyme A were 0.65 and 7.4 mM, respectively. The enzyme was at a low level in log-phase cells but increased up to 10-fold early in the stationary phase and had a similar specific activity in both the mother cell and the forespore compartment; the enzyme activity fell only slowly during spore germination and outgrowth. The enzyme was not detected in several eucaryotic sources and was present in at most a low level in a number of gram-negative bacteria. Surprisingly, the specific activity of this enzyme varied more than 200-fold in extracts from different Bacillus species, with values in B. subtilis being 5- to 6-fold lower and values in B. cereus and B. sphaericus being 8- and 35-fold higher, respectively, than the maximum value in B. megaterium. However, the high specific activity in B. sphaericus did not represent more enzyme protein than in B. megaterium. The possible function of this newly discovered enzyme is discussed.
D001407:Bacillus / Q000201:enzymology; D001410:Bacillus megaterium / Q000201:enzymology* / Q000502:physiology; D004220:Disulfides / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010204:Pantetheine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D013171:Spores, Bacterial / Q000201:enzymology; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; C020631:pantethine 4',4''-diphosphate; D010204:Pantetheine; D009247:NADH, NADPH Oxidoreductases; C014703:disulfide reductase (NADH)