Structural characterization of CYP165D3, a cytochrome P450 involved in phenolic coupling in teicoplanin biosynthesis
Structural characterization of CYP165D3, a cytochrome P450 involved in phenolic coupling in teicoplanin biosynthesis
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DOI：
10.1016/j.abb.2010.10.017
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发表时间：
2011-03-01
影响因子：
3.9
通讯作者：
Schlichting, Ilme
中科院分区：
文献类型：
--
作者：
Cryle, Max J.;Staaden, Jessica;Schlichting, Ilme
替考拉宁是一种糖肽类抗生素，对革兰氏阳性菌具有活性，仍然是临床上抵抗某些细菌感染的最后防线之一。我们从替考拉宁生物合成基因簇中克隆、表达并纯化了细胞色素P450 OxyE（CYP 165 D3），该基因簇负责替考拉宁肽中第一和第三个肽残基芳香族侧链的酚偶联。OxyE的晶体结构已被确定为2.5埃分辨率，揭示了载体蛋白底物的可能结合表面和活性位点延伸到位于β-1片层上方的口袋中。潜在底物与OxyE的结合表明，肽基载体蛋白结合的线性肽与OxyE结合，尽管在不存在酚交联的情况下亲和力较低，而酚交联通常应由替考拉宁生物合成途径中的另一种Oxy蛋白安装。该结果表明，单独的载体蛋白不足以使底物与OxyE紧密结合，并且除了载体蛋白的存在之外，Oxy蛋白还感测结合肽的结构，这是不同于其它载体蛋白/P450系统的特征。(C)2010年爱思唯尔公司All rights reserved.
Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned, expressed, and purified the cytochrome P450 OxyE (CYP165D3) from the teicoplanin biosynthetic gene cluster of Actinoplanes teichomyceticus, which is responsible for the phenolic coupling of the aromatic side chains of the first and third peptide residues in the teicoplanin peptide. The crystal structure of OxyE has been determined to 2.5 angstrom resolution, revealing the probable binding surface for the carrier protein substrate and an extension of the active site into a pocket located above the beta-1 sheet. The binding of potential substrates to OxyE shows that peptidyl carrier protein-bound linear peptides bind to OxyE, albeit with low affinity in the absence of a phenolic cross-link that should normally be installed by another Oxy protein in the teicoplanin biosynthetic pathway. This result indicates that the carrier protein alone is not sufficient for tight substrate binding to OxyE and that the Oxy proteins sense the structure of the bound peptide in addition to the presence of the carrier protein, a feature distinct from other carrier protein/P450 systems. (C) 2010 Elsevier Inc. All rights reserved.