Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin
Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin
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DOI：
10.1021/bi981699c
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发表时间：
1998-10-20
期刊：
影响因子：
2.9
通讯作者：
McDaniel, R
中科院分区：
文献类型：
--
作者：
Betlach, MC;Kealey, JT;McDaniel, R
大环内酯类抗生素的聚酮后合成酶（PKS）生物合成剪裁通常涉及细胞色素P450单加氧酶催化的一个或多个氧化反应。由于这类酶的成员在不同PKS基因簇中的特异性差异很大，因此鉴定和研究新的大环内酯类p450对组合生物合成领域的发展具有重要意义。我们从委内瑞拉链霉菌中分离出细胞色素P450基因picK，该基因负责将纳博霉素的C-12羟基化为微霉素。用大环内酯P450单加氧酶探针搜索模块化PKS基因附近的区域，确定了该基因的位置。在大肠杆菌中过量产生带有c端6- his亲和标签（PicK/6-Kis）的PicK有助于酶的纯化以进行动力学分析。结果表明，PicK/6-Kis在体外催化纳博霉素的C-12羟基化，k（cat）为1.4 s(-1)，这与EryK羟化酶对红霉素D的C-12羟基化的报道值相似。该酶的独特特异性应该有助于修饰类似纳博霉素的新型大环内酯底物，特别是酮内酯，一类有前途的半合成大环内酯类，具有抗红霉素耐药病原体的活性。
The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to the growing field of combinatorial biosynthesis. We have isolated the cytochrome P450 gene picK from Streptomyces venezuelae which is responsible for the C-12 hydroxylation of narbomycin to picromycin. The gene was located by searching regions proximal to modular PKS genes with a probe for macrolide P450 monooxygenases. The overproduction of PicK with a C-terminal six-His affinity tag (PicK/6-Kis) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-Kis was shown to catalyze the in vitro C-12 hydroxylation of narbomycin with a k(cat) of 1.4 s(-1), which is similar to the value reported for the related C-12 hydroxylation of erythromycin D by the EryK hydroxylase. The unique specificity of this enzyme should be useful for the modification of novel macrolide substrates similar to narbomycin, in particular, ketolides, a promising class of semisynthetic macrolides with activity against erythromycin-resistant pathogens.