Connors N C, Strohl W R
Department of Microbiology, Ohio State University, Columbus 43210.
J Gen Microbiol. 1993 Jun;139 Pt 6:1353-62. doi: 10.1099/00221287-139-6-1353.
A methyltransferase that acts on carminomycin and 13-dihydrocarminomycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycin- and baumycin-producing Streptomyces sp. strain C5. The enzyme was obtained in approximately 5% yield with a purification of 114-fold in specific activity over the sample precipitated with 30-50% ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit M(r) of about 41,000. The enzyme was shown by gel filtration chromatography to have an M(r) of approximately 166,000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for S-adenosyl-L-methionine typical of antibiotic methyltransferases; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. S-Adenosyl-L-homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, epsilon-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.
一种作用于洋红霉素和13 - 二氢洋红霉素的甲基转移酶，据推测它是柔红霉素生物合成途径中的末端酶，已从产生柔红霉素和鲍霉素的链霉菌属菌株C5中纯化至近乎同质。该酶的产率约为5%，比用30 - 50%硫酸铵沉淀的样品的比活性纯化了114倍。变性条件下的聚丙烯酰胺凝胶电泳表明亚基分子量约为41,000。凝胶过滤色谱显示该酶的分子量约为166,000，表明它是同四聚体。动力学分析表明其对S - 腺苷 - L - 甲硫氨酸具有抗生素甲基转移酶典型的亲和力；该酶对洋红霉素的亲和力略高于对13 - 二氢洋红霉素的亲和力。通过色谱和质谱分析证实，洋红霉素甲基化的反应产物是柔红霉素。纯化的酶不催化糖苷配基洋红霉素酮或13 - 二氢洋红霉素酮的甲基化。S - 腺苷 - L - 高半胱氨酸抑制甲基转移酶，而高半胱氨酸、腺苷、腺嘌呤、ε - 红霉酮、柔红霉素和柔红霉素酮几乎没有抑制活性或没有抑制活性。
