PURIFICATION AND CHARACTERIZATION OF VIRGINIAMYCIN-M-1 REDUCTASE FROMSTREPTOMYCES-VIRGINIAE

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.