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首页> 外文期刊>Biomolecular engineering >Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals.
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Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals.

机译：通过定向进化设计细胞色素P450 CYP102A2的底物特异性：生产用于精细化学品生物转化的有效酶。

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摘要

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and varepsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.
机译：P450细胞色素构成大量的血蛋白，可催化多种疏水性底物的单加氧作用。 CYP102A2是一种来自枯草芽孢杆菌的催化自给自足的细胞质酶，在一条多肽链上同时包含单加氧酶结构域和还原酶结构域。对CYP102A2进行易错PCR反应，以生成具有增强的脂肪酸和其他芳香族底物活性的突变体。 CYP102A2突变体的文库在BL21（DE3）大肠杆菌细胞中表达，并通过活性测定筛选其氧化不同底物的能力。在单轮易错PCR之后，生成了显示修饰的底物特异性的变体Pro15Ser。该变体显示出对SDS，月桂酸和1,4-萘醌的活性提高了约6到9倍，并且对其他底物（如乙炔酸和缬草胺-氨基-正己酸）的活性增强。 CYP102A2单加氧酶域的分子模型表明，Pro15位于短螺旋段中，并参与N末端域和beta2折叠之间的广泛相互作用，这有助于底物结合位点的形成。因此，Pro15似乎间接影响底物的结合和催化。这些结果清楚地证明了通过合理的设计无法轻易预测的远程残基对于确定底物特异性的重要性。此外，我们在此报告CYP102A2的Pro15Ser变体可以在pH 8.5和4摄氏度下有效地固定在环氧活化的琼脂糖上。CYP102A2的固定变体保留了其大部分活性（81％），并在37度下显示出改善的稳定性C.该方法提供了设计P450生物反应器的可能性，该P450生物反应器可以长时间高效运行并且可以用于精细化学合成中。

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