Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae
Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae
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DOI：
10.1016/s1074-5521(98)90293-9
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发表时间：
1998-11-01
影响因子：
--
通讯作者：
Sherman, DH
中科院分区：
文献类型：
--
作者：
Xue, YQ;Wilson, D;Sherman, DH
背景资料：委内瑞拉链霉菌产生两组抗生素，包括12元环大环内酯甲霉素和新甲霉素，以及14元环大环内酯吡克罗霉素。甲霉素和吡克罗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）的羟基化衍生而来。相比之下，新甲基霉素是由YC-17的丙酰基起始单元sidechain.Results的仲碳（C-12）的羟基化衍生而来的：使用遗传和生化方法，我们已经确定了一个单一的P450羟化酶（PikC）的甲基霉素/匹克罗霉素生物合成基因簇（Pik）从S。委内瑞拉。pikC的失活消除了所有羟基化大环内酯类的产生，在培养基中相应地积累了YC-17和纳博霉素。该酶被有效地生产和纯化为His-标记的蛋白质从重组大肠杆菌细胞。纯化的PikC在体外可有效地将YC-17转化为甲基霉素和新甲基霉素，将纳博霉素转化为吡克霉素。委内瑞拉。这种底物的灵活性是独特的，代表了P450羟化酶的第一个例子，它可以接受12-和14-元环大环内酯作为底物，以及在大环内酯系统上的两个位置官能化。PikC广泛的底物特异性为构建新型大环内酯和酮内酯类抗生素提供了潜在的有价值的入口。
Background: 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.Results: 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.Conclusions: 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.