Crystal structure of OxyC, a cytochrome P450 implicated in an oxidative C-C coupling reaction during vancomycin biosynthesis
Crystal structure of OxyC, a cytochrome P450 implicated in an oxidative C-C coupling reaction during vancomycin biosynthesis
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DOI：
10.1074/jbc.m306486200
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发表时间：
2003-11-21
影响因子：
4.8
通讯作者：
Schlichting, I
中科院分区：
文献类型：
--
作者：
Pylypenko, O;Vitali, F;Schlichting, I
基因失活研究指出OxyC参与催化糖肽抗生素生物合成过程中的最后一个氧化苯酚偶联反应。目前，OxyC反应的底物和确切时间是未知的。底物可以是与蛋白质载体结构域结合的双环七肽或硫酯衍生物。在大肠杆菌中产生来自万古霉素生产者东方拟无枝酸菌的OxyC并结晶，并确定其结构为1.9埃分辨率。OxyC给出了低自旋铁态的P450样血红素蛋白的紫外-可见光谱特征。通过连二亚硫酸盐还原成亚铁态后，CO-连接形式在UV差光谱中产生450 nm峰。向OxyC中加入万古霉素糖苷配基产生了UV光谱的I型变化。OxyC表现出典型的P450倍，其中Cys配体环含有特征序列FGHGX-HXCLG，Cys-356是血红素铁的近端轴向硫醇配体。与血红素铁结合的水分子的观察结果与表明低自旋血红素的OxyC的UV-可见光谱一致。占据活性位点的聚乙二醇分子可能模拟双环七肽底物。氧蛋白和其他P450的结构分析表明，可能参与结合的氧化还原伙伴和可能的蛋白载体结构域的区域。
Gene inactivation studies point to the involvement of OxyC in catalyzing the last oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. Presently, the substrate and exact timing of the OxyC reaction are unknown. The substrate might be the bicyclic heptapeptide or a thioester derivative bound to a protein carrier domain. OxyC from the vancomycin producer Amycolatopsis orientalis was produced in Escherichia coli and crystallized, and its structure was determined to 1.9 Angstrom resolution. OxyC gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. The addition of vancomycin aglycone to OxyC produced type I changes to the UV spectrum. OxyC exhibits the typical P450-fold, with the Cys ligand loop containing the signature sequence FGHGX-HXCLG and Cys-356 being the proximal axial thiolate ligand of the heme iron. The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme. A polyethylene glycol molecule occupying the active site might mimic the bicyclic heptapeptide substrate. Analysis of the structure of Oxy-proteins and other P450s indicates regions that might be involved in binding of the redox partner and possibly the protein carrier domain.