Hydrogen peroxide dependent monooxygenations by tricking the substrate recognition of cytochrome P450BSβ
Hydrogen peroxide dependent monooxygenations by tricking the substrate recognition of cytochrome P450BSβ
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DOI：
10.1002/anie.200700068
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Watanabe, Yoshihito
中科院分区：
文献类型：
--
作者：
Shoji, Osami;Fujishiro, Takashi;Watanabe, Yoshihito
The design and construction of enzymes as biocatalysts has been the subject of intensive studies because of their high regio-and enantioselectivities as well as high activities.[1, 2] For example, site-directed mutagenesis and random mutagenesis are powerful tools to transform native enzymes into biocatalysts that are applicable to industrial processes. The construction of protein active sites by rational design is a good example of site-directed mutagenesis.[3] Although the mutation sites in the random mutagenesis (so-called “directed evolution”) are rather unpredictable, effective substitution of amino acids to introduce desired functions has been demonstrated.[4, 5] Cytochrome P450s (P450s), which are hemecontaining monooxygenases, play important roles in drug metabolism, detoxification of xenobiotics, and steroid biosynthesis. In these reactions, P450s catalyze hydroxylation of less reactive CÀH bonds, a key reaction in organic synthesis.[6, 7] A series of P450 mutants have been prepared to alter their substrate specificity and improve catalytic activity.[8–10] Selective oxidation of saturated CÀH bonds by biocatalysts is of great importance because of their potential use in industrial applications.[11, 12] Alkane hydroxylation by engineered P450cam [13] and P450 BM-3 [8] has been reported, and Meinhold et al. and Xu et al. have recently prepared P450 mutants to catalyze ethane hydroxylation at relatively high turnover rates under mild conditions.[14, 15] Unfortunately, the practical use of these P450s is limited, since they require the very expensive electron-donating cofactor NAD (P) H for the reductive oxygen activation. Although NAD (P) H-regeneration systems effectively reduce the cost, the catalytic systems become complicated and thus have other limitations.[16] Instead of the cofactors and molecular oxygen, P450s are capable of accepting peroxides as an oxidant; this is the socalled peroxide-shunt pathway. However, P450 systems using hydrogen peroxide (H2O2) are inefficient and impractical.[17, 18]In contrast to most P450s, P450BSβ (CYP152A1), isolated from Bacillus subtilis, efficiently utilizes H2O2 to catalyze the exclusive hydroxylation of long-alkyl-chain fatty acids such as myristic acid to give β-hydroxymyristic acid (60%) and αhydroxymyristic acid (40%).[19, 20] This soluble enzyme does not require any cofactors, including electron-transfer systems; it works as a single component. Because of its low cost, H2O2 can be used as an oxidant in industrial-scale processes, and P450BSβ would thus be a great candidate for practical biocatalysts. The X-ray crystal structure analysis of the palmitic acid bound form of P450BSβ[20] suggests a unique catalytic mechanism: 1) The catalytic reaction begins with the fixation of a substrate through interaction of the terminal carboxy group of the fatty acid with Arg242, located near the heme; 2) A general acid–base function of the fatty acid–Arg242 salt bridge allows facile generation of the active species, the so-called Compound I,[21, 22] to oxidize the substrate (Figure 1).[23] Without the interaction of the carboxy group, P450BSβ does not accept H2O2 to start its reaction. Therefore, the fatty acid substrate itself is the initiator as well as the activator of the reaction. As this unique catalytic mechanism contributes to high substrate specificity and regioselectivity of the hydroxylation,[19] P450BSβ never oxidizes substrates other than fatty acids that have long alkyl chains. If one could trick P450BSβ by using a decoy molecule with a carboxy group which is recognized by P450BSβ as a substrate to make the carboxylate–Arg salt bridge but is not oxidized by P450BSβ, a wide variety of nonnatural substrates could …
