Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis
Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis
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DOI：
10.1007/s00775-010-0692-4
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发表时间：
2010-08
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影响因子：
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通讯作者：
O. Shoji;T. Fujishiro;S. Nagano;Shota Tanaka;T. Hirose;Y. Shiro;Y. Watanabe
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文献类型：
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作者：
O. Shoji;T. Fujishiro;S. Nagano;Shota Tanaka;T. Hirose;Y. Shiro;Y. Watanabe
细胞色素P450 BS β是一种H2 O2依赖性的细胞色素P450，催化长烷基链脂肪酸的羟基化，缺乏血红素周围的一般酸碱残基，这是使用H2 O2有效产生活性物种所必需的。根据细胞色素P450 BS β的棕榈酸结合形式的晶体结构，提出了一般酸碱功能的作用是由脂肪酸的羧酸基团提供的。底物的羧酸基团的参与是由细胞色素P450 BS β在一系列短链烷基羧酸作为脂肪酸的虚拟分子存在下催化非天然底物如苯乙烯和苯乙烯的氧化这一事实支持的。我们将一系列短烷基链羧酸称为“诱饵分子”。如这里所示，我们已经澄清了诱饵分子结合形式的晶体结构，并阐明其羧酸根基团的位置与血红素腔中棕榈酸的位置几乎相同，表明诱饵分子的羧酸根基团充当一般的酸碱催化剂。这一结果进一步证实了酸-碱官能团的作用由底物的羧酸根基团满足。此外，无底物形式的结构分析已经阐明，诱饵分子以及脂肪酸的结合没有引起显着的结构变化。因此，羧酸基团是否位于活性位点提供了细胞色素P450 BS β催化循环的切换机制。
Cytochrome P450BSβ, a H2O2-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid–base residue around the heme, which is indispensable for the efficient generation of the active species using H2O2. On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450BSβ, it was suggested that the role of the general acid–base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450BSβcan catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a “decoy molecule”. As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid–base catalyst. This result further confirms that the role of the acid–base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450BSβ.