A substrate-binding-state mimic of H2O2-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates
A substrate-binding-state mimic of H2O2-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates
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DOI：
10.1039/c6cy00630b
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发表时间：
2016-07
影响因子：
5
通讯作者：
O. Shoji;T. Fujishiro;K. Nishio;Y. Kano;H. Kimoto;S. Chien;Hiroki Onoda;A. Muramatsu;Shota Tanaka-Sh
中科院分区：
文献类型：
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作者：
O. Shoji;T. Fujishiro;K. Nishio;Y. Kano;H. Kimoto;S. Chien;Hiroki Onoda;A. Muramatsu;Shota Tanaka-Sh
通过 P450SPα (CYP152B1) 的单点诱变构建了 H2O2 依赖性细胞色素 P450 的底物结合态模拟物，能够催化非天然底物的单氧化作用。 P450SPα 是一种长烷基链脂肪酸羟化酶，血红素周围缺乏任何一般酸碱残基。因此，脂肪酸的羧基对于使用 H2O2 生成活性物质是必不可少的。我们制备了 A245E 突变体，通过在活性位点放置羧酸根来模拟底物结合状态。 A245E 突变体的活性位点结构与 P450SPα 的脂肪酸结合状态相似，并以 280 min−1 (kcat) 的速率催化苯乙烯氧化，而野生型酶不显示任何催化活性。更重要的是，相同的突变，即P450中高度保守的苏氨酸突变为谷氨酸，也能有效地将过氧合酶活性引入P450BM3、P450cam和CYP119中。这些结果表明，可以通过单点诱变构建多种基于P450的过氧化酶。
A substrate-binding-state mimic of H2O2-dependent cytochrome P450 that is able to catalyze monooxygenation of non-native substrates was constructed by one-point mutagenesis of P450SPα (CYP152B1). P450SPα, a long-alkyl-chain fatty acid hydroxylase, lacks any general acid–base residue around the heme. The carboxylate group of a fatty acid is thus indispensable for the generation of active species using H2O2. We prepared an A245E mutant to mimic a substrate-binding state by placing a carboxylate group at the active site. The active site structure of the A245E mutant is similar to that of the fatty-acid-bound state of P450SPα and catalyzes styrene oxidation at a rate of 280 min−1 (kcat), whereas the wild-type enzyme does not show any catalytic activity. More importantly, the same mutation, i.e. the mutation of the highly conserved threonine in P450s to glutamic acid, was also effective in introducing peroxygenase activity into P450BM3, P450cam, and CYP119. These results indicate that a variety of peroxygenases based on P450s can be constructed by one-point mutagenesis.