Suzuki N, Lee C K, Nihira T, Yamada Y
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
Antimicrob Agents Chemother. 1998 Nov;42(11):2985-8. doi: 10.1128/AAC.42.11.2985.
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.
弗吉尼亚霉素M1（VM1）由弗吉尼亚链霉菌产生，是一种属于弗吉尼亚霉素A组的多不饱和大环内酯类抗生素。弗吉尼亚链霉菌具有一种活性，可立体特异性地还原VM1的16-羰基，产生无抗菌活性的16R-二氢VM1。通过五步从弗吉尼亚链霉菌的粗提取物中纯化得到了相应的VM1还原酶，纯化倍数为5650倍，总产率为23%。测定其N端氨基酸序列为MAIKLVIA。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，纯化后的酶表观分子量为73000，通过天然分子筛高效液相色谱法测定其分子量为280000，表明该天然酶具有四聚体性质。NADPH作为还原反应的辅酶，Km值为0.13 mM，但NADH即使在浓度为5 mM时也不支持该反应，表明该酶具有NADPH特异性。在2 mM NADPH存在下，VM1的Km值为1.5 mM。在逆向反应中，只有16R-二氢VM1，而不是16S-差向异构体，作为底物，与正向反应相比，总反应速率小于0.1%，证实VM1还原酶在体内仅参与VM1的失活过程。
