N. Suzuki et al., PURIFICATION AND CHARACTERIZATION OF VIRGINIAMYCIN-M-1 REDUCTASE FROMSTREPTOMYCES-VIRGINIAE, Antimicrobial agents and chemotherapy, 42(11), 1998, pp. 2985-2988
Virginiamycin M-1 (VM1), produced by Streptomyces virginiae, is a poly
unsaturated macrocyclic lactone antibiotic belonging to the virginiamy
cin A group. S. virginiae possesses an activity which stereospecifical
ly reduces a 16-carbonyl group of VM1, resulting in antibiotically ina
ctive 16R-dihydroVM(1). The corresponding VM1 reductase was purified t
o homogeneity from crude extracts of S. virginiae in five steps, with
5,650-fold purification and 23% overall yield. The N-terminal amino ac
id sequence was determined to be MAIKLVIA. The purified enzyme showed
an apparent M-r of 73,000 by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and an M-r 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 K-m value of 0.13 mM, but NADH did not support the reaction, even a
t a concentration of 5 mM, indicating the NADPH-specific nature of the
enzyme. The K-m for VM1 was determined to be 1.5 mM in the presence o
f 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM(1), not the
16S-epimer, served as a substrate, with a less than 0.1% overall react
ion rate compared to that of the forward reaction, confirming that the
VM1 reductase participates solely in VM1 inactivation in vivo.