Xue Y, Wilson D, Zhao L, Liu H w, Sherman D H
Department of Microbiology Biological Process Technology Institute University of Minnesota Minneapolis MN 55455 USA.
Chem Biol. 1998 Nov;5(11):661-7. doi: 10.1016/s1074-5521(98)90293-9.
. Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin. Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring. In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain.
. Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S. venezuelae. Inactivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium. The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coli cells. Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro.
. These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.
委内瑞拉链霉菌产生两类抗生素，包括12元环大环内酯类的美伐霉素和新美伐霉素，以及14元环大环内酯类的苦霉素。美伐霉素和苦霉素分别由相应的前体YC - 17和纳博霉素通过大环内酯环上叔碳位置（YC - 17中的C - 10或纳博霉素中的C - 12）的羟基化作用衍生而来。相比之下，新美伐霉素是由YC - 17通过丙酰基起始单元侧链仲碳（C - 12）的羟基化作用衍生而来。
我们采用遗传和生化方法，对委内瑞拉链霉菌中甲霉素/苦霉素生物合成基因簇（pik）中的一种单一细胞色素P450羟化酶（PikC）进行了表征。pikC的失活消除了所有羟基化大环内酯类抗生素的产生，培养基中相应积累了YC - 17和纳博霉素。该酶在重组大肠杆菌细胞中高效表达，并作为His标签蛋白进行纯化。纯化后的PikC在体外能有效地将YC - 17转化为美伐霉素和新美伐霉素，将纳博霉素转化为苦霉素。
这些结果表明，PikC负责委内瑞拉链霉菌中YC - 17向美伐霉素和新美伐霉素以及纳博霉素向苦霉素的转化。这种底物灵活性是独特的，代表了一种细胞色素P450羟化酶的首个实例，该酶能够接受12元和14元环大环内酯类作为底物，并能在大环内酯系统的两个位置进行官能化。PikC广泛的底物特异性为构建新型大环内酯类和酮内酯类抗生素提供了潜在的有价值途径。
