Structural Fine-Tuning of a Multifunctional Cytochrome P450 Monooxygenase
Structural Fine-Tuning of a Multifunctional Cytochrome P450 Monooxygenase
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DOI：
10.1021/ja110146z
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发表时间：
2011-02-23
影响因子：
15
通讯作者：
Hertweck, Christian
中科院分区：
文献类型：
--
作者：
Zocher, Georg;Richter, Martin E. A.;Hertweck, Christian
AurH是一种独特的细胞色素P450单加氧酶，催化同手性氧杂环的逐步形成，该杂环是抗生素金黄色的关键结构和药理作用成分。这种特殊的酶反应包括一个串联的氧化过程，包括区域和立体特异的羟基化，然后是杂环化。对于这一序列的结构和生化基础，我们解决了AurH变异体在不同构象状态和与P450抑制剂ancymidol的络合物中的四个晶体结构，这是CYP151a基团的第一个结构。结构数据与计算对接、定点突变和化学分析相结合，揭示了在识别两种底物-脱氧金黄和羟化中间体时的开关功能，从而允许第二氧化-杂环化步骤。此外，我们能够改变AurH的化学和区域选择性，产生突变体，通过羟基和醛中间体催化未激活的甲基到羧酸的区域选择性六电子转移。
AurH is a unique cytochrome P450 monooxygenase catalyzing the step-wise formation of a homochiral oxygen heterocycle, a key structural and pharmacophoric component of the antibiotic aureothin. The exceptional enzymatic reaction involves a tandem oxygenation process including a regio- and stereospecific hydroxylation, followed by heterocyclization. For the structural and biochemical basis of this unparalleled sequence, four crystal structures of AurH variants in different conformational states and in complex with the P450 inhibitor ancymidol were solved, which represent the first structures of the CYP151A group. Structural data in conjunction with computational docking, site-directed mutagenesis, and chemical analyses unveiled a switch function when recognizing the two substrates, deoxyaureothin and the hydroxylated intermediate, thus allowing the second oxygenation-heterocyclization step. Furthermore, we were able to modify the chemo- and regioselectivity of AurH, yielding mutants that catalyze the regioselective six-electron transfer of a nonactivated methyl group to a carboxylic acid via hydroxyl and aldehyde intermediates.