Purification and characterization of virginiamycin M1 reductase from Streptomyces virginiae.
Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrocyclic lactone antibiotic belonging to the virginiamycin A group. S. virginiae possesses an activity which stereospecifically reduces a 16-carbonyl group of VM1, resulting in antibiotically inactive 16R-dihydroVM1. The corresponding VM1 reductase was purified to homogeneity from crude extracts of S. virginiae in five steps, with 5,650-fold purification and 23% overall yield. The N-terminal amino acid sequence was determined to be MAIKLVIA. The purified enzyme showed an apparent Mr of 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 280,000 by native molecular sieve high-performance liquid chromatography, indicating the tetrameric nature of the native enzyme. NADPH served as a coenzyme for the reduction, with a Km value of 0.13 mM, but NADH did not support the reaction, even at a concentration of 5 mM, indicating the NADPH-specific nature of the enzyme. The Km for VM1 was determined to be 1.5 mM in the presence of 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM1, not the 16S-epimer, served as a substrate, with a less than 0.1% overall reaction rate compared to that of the forward reaction, confirming that the VM1 reductase participates solely in VM1 inactivation in vivo.
Suzuki
N
N
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
Lee
C K
CK
Nihira
T
T
Yamada
Y
Y
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D013302:Streptomyces / Q000201:enzymology*; D014769:Virginiamycin / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D014769:Virginiamycin; D009249:NADP; D010088:Oxidoreductases