The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae
The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae
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DOI：
10.1074/jbc.m605478200
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发表时间：
2006-09-08
影响因子：
4.8
通讯作者：
Podust, Larissa M.
中科院分区：
文献类型：
--
作者：
Sherman, David H.;Li, Shengying;Podust, Larissa M.
维尼链霉菌(Streptomyces Venezuelae)的匹克罗霉素(Pik)/甲氧霉素生物合成途径是研究聚酮模块生物合成、氨基脱氧糖组装、糖基转移和羟基化等一系列大环内酯类抗生素产生的重要途径。在这项研究中，我们描述了PIKC的四种X射线晶体结构和相关的功能研究，PIKC是一种非凡的P450单加氧酶，负责从PIK途径产生许多相关的大环内酯类产物。这些结果为12-(YC-17)和14元环(那宝霉素)大环内酯类化合物的C10/C12和C12/C14羟基化模式的结构基础提供了重要的新见解。这包括一个不对称单元中的两个不同的无配体结构(分辨率2.1埃)和两个具有结合内源底物YC-17(分辨率2.35埃)或那宝霉素(分辨率1.7埃)的共晶结构。酶-底物相互作用的一个主要特征是将脱糖胺残基锚定在两个可供选择的结合口袋中，这两个结合口袋基于一系列不同的氨基酸残基，形成盐桥和与脱氧糖C3‘二甲氨基基的氢键网络。点突变证实了盐桥的功能意义，揭示了Glu-94在YC-17结合中的关键作用，Glu-85在那宝霉素结合中起关键作用。综上所述，X射线结构分析、定点突变和相应的产物分布研究表明，PIKC底物耐受性和产物多样性是替代锚定模式的组合而不是诱导FIT机制的结果。
The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 angstrom) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 angstrom) or narbomycin (resolution 1.7 angstrom). A central feature of the enzyme-substrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3' dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.