Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton
Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton
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DOI：
10.1073/pnas.0702946104
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发表时间：
2007-07-10
影响因子：
11.1
通讯作者：
Nagano, Shingo
中科院分区：
文献类型：
--
作者：
Makino, Masatomo;Sugimoto, Hiroshi;Nagano, Shingo
从链霉菌属中分离出星形孢菌素。 TP-A0274 是吲哚并咔唑生物碱家族的成员，具有很强的抗肿瘤活性。星形孢菌素生物合成的关键步骤是通过分子内 C-C 键形成和细胞色素 P450 StaP（StaP，CYP245A1）催化的铬吡咯酸（CPA）氧化脱羧形成吲哚并咔唑核心。在这项研究中，我们报告了结合 CPA 和游离形式的 StaP 的 X 射线晶体结构。与底物结合后，StaP 采用更有序的构象，并且还观察到活性位点残基的构象重排。两个羧基的氢键相互作用以及与吲哚环的 T 形 pi-pi 相互作用将底物保持在底物结合腔中，并具有垂直于血红素平面的构象。基于StaP-CPA复合物的晶体结构，我们提出C-C键的形成是通过相当于细胞色素c过氧化物酶化合物I的吲哚阳离子自由基中间体发生的[Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738-7401。还根据晶体结构讨论了随后的氧化脱羧反应。我们的晶体学研究显示了参与吲哚并咔唑核心形成的酶的第一个晶体结构，并为星形孢菌素生物合成过程、吲哚并咔唑的组合生物合成以及细胞色素 P450 化学的多样性提供了宝贵的见解。
Staurosporine isolated from Streptomyces sp. TP-A0274 is a member of the family of indolocarbazole alkaloids that exhibit strong antitumor activity. A key step in staurosporine biosynthesis is the formation of the indolocarbazole core by intramolecular C-C bond formation and oxidative decarboxylation of chromopyrrolic acid (CPA) catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we report x-ray crystal structures of CPA-bound and -free forms of StaP. Upon substrate binding, StaP adopts a more ordered conformation, and conformational rearrangements of residues in the active site are also observed. Hydrogen-bonding interactions of two carboxyl groups and T-shaped pi-pi interactions with indole rings hold the substrate in the substrate-binding cavity with a conformation perpendicular to the heme plane. Based on the crystal structure of StaP-CPA complex, we propose that C-C bond formation occurs through an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I [Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738-7401. The subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.