Theoretical and experimental studies of the conversion of chromopyrrolic acid to an antitumor derivative by cytochrome P450 StaP: the catalytic role of water molecules.
Theoretical and experimental studies of the conversion of chromopyrrolic acid to an antitumor derivative by cytochrome P450 StaP: the catalytic role of water molecules.
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DOI：
10.1021/ja9003365
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发表时间：
2009-04
影响因子：
15
通讯作者：
Yong Wang;Hui Chen;M. Makino;Y. Shiro;S. Nagano;Shumpei Asamizu;H. Onaka;S. Shaik
中科院分区：
文献类型：
--
作者：
Yong Wang;Hui Chen;M. Makino;Y. Shiro;S. Nagano;Shumpei Asamizu;H. Onaka;S. Shaik
通过细胞色素P450 StaP氧化色吡咯酸（CPA）是抗肿瘤药物生物合成中的关键过程（Onaka，H.; Taniguchi，S.; Igarashi，Y.; Furumai，T. Biosci. Biotechnol. 2003，67，127-138），其通过不寻常的C-C键偶联进行。此外，由于CPA通过氢键阵列固定，因此它不能与P450的活性物质化合物I直接反应。因此，P450 StaP的机制构成了一个谜。在本文中，我们解决了这个难题的理论相结合，使用QM/MM计算，和实验，使用晶体学和反应性研究。理论表明，口袋的氢键机制使CPA的羧酸基团去质子化，而附近的His（250）残基和晶体沃茨，Wat（644）和Wat（789），帮助双去质子化的CPA将电子密度转移到化合物I;因此，CPA被活化朝向质子偶联电子转移，使整个机制处于运动状态。随后的机制涉及C-C键形成的步骤耦合到第二个电子转移，四个质子转移和互变异构化步骤，和四个步骤，其中Wat（644）和Wat（789）移动和介导这些事件。用二氯化底物CCA进行的实验表明，这种酶失去了活性，CCA会排出瓦特（644）。P450 StaP的H250 A和H250 F突变表明His（250）是重要的，但在其缺失时，Wat（644）和Wat（789）形成介导转化的氢键二联体。因此，水二分体作为赋予StaP功能的最小必要元素而出现。这突出了水分子作为将P450转化为过氧化物酶型的生物催化剂的作用（Derat，E.;谢克，S。J. Am. 2006，128，13940-13949）。
Chromopyrrolic acid (CPA) oxidation by cytochrome P450 StaP is a key process in the biosynthesis of antitumor drugs (Onaka, H.; Taniguchi, S.; Igarashi, Y.; Furumai, T. Biosci. Biotechnol. Biochem. 2003, 67, 127-138), which proceeds by an unusual C-C bond coupling. Additionally, because CPA is immobilized by a hydrogen-bonding array, it is prohibited from undergoing direct reaction with Compound I, the active species of P450. As such, the mechanism of P450 StaP poses a puzzle. In the present Article, we resolve this puzzle by combination of theory, using QM/MM calculations, and experiment, using crystallography and reactivity studies. Theory shows that the hydrogen-bonding machinery of the pocket deprotonates the carboxylic acid groups of CPA, while the nearby His(250) residue and the crystal waters, Wat(644) and Wat(789), assist the doubly deprotonated CPA to transfer electron density to Compound I; hence, CPA is activated toward proton-coupled electron transfer that sets the entire mechanism in motion. The ensuing mechanism involves a step of C-C bond formation coupled to a second electron transfer, four proton-transfer and tautomerization steps, and four steps where Wat(644) and Wat(789) move about and mediate these events. Experiments with the dichlorinated substrate, CCA, which expels Wat(644), show that the enzyme loses its activity. H250A and H250F mutations of P450 StaP show that His(250) is important, but in its absence Wat(644) and Wat(789) form a hydrogen-bonding diad that mediates the transformation. Thus, the water diad emerges as the minimal requisite element that endows StaP with function. This highlights the role of water molecules as biological catalysts that transform a P450 to a peroxidase-type (Derat, E.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 13940-13949).