Engineered alkane-hydroxylating cytochrome P450BM3 exhibiting nativelike catalytic properties
Engineered alkane-hydroxylating cytochrome P450BM3 exhibiting nativelike catalytic properties
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DOI：
10.1002/anie.200702616
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
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作者：
Fasan, Rudi;Chen, Mike M.;Arnold, Frances H.
细胞色素P450酶（P450）是特殊的氧化催化剂[1]，在药物发现，化学合成，生物修复和生物技术方面具有巨大的潜力。[2，3]然而，与它们的天然对应物相比，工程化的P450通常表现出较差的催化和辅因子偶联效率。[3]获得类似天然的催化活性是在化学合成中利用这些多功能加氧酶的能力的必要的第一步。细胞色素P450 BM 3（119 kDa，B. megaterium）催化长链（C12-C20）脂肪酸的亚末端羟基化。[4]它的高活性和催化自给自足（血红素和二黄素还原酶结构域融合在一条多肽链中）[2，4，5]使P450 BM 3成为生物催化的优良平台。然而，尽管有许多报道称血红素结构域被改造成接受非天然底物，包括短链脂肪酸、芳香族化合物、烷烃和烯烃，[6-8]但P450 BM 3的大规模应用的报道仍然很少。[9]P450 BM 3功能通过血红素和还原酶结构域中的构象重排以及可能还通过铰链结构域运动进行精细调节。[4，10]脂肪酸的羟基化几乎完全与辅因子（NADPH）的利用（93-96%，取决于底物）相结合。[11]在存在非天然底物或引入氨基酸取代时，控制P450有效催化的机制被破坏，[12]导致活性氧的形成和快速酶失活。[4]尚未实现在其物理化学性质与天然底物显著不同的底物上的高偶联效率，并且偶联效率范围为小于1%至30-40%是典型的。[7，8]为了将工程化的P450应用于更大规模的应用，需要解决这种“偶联问题”的策略。短链烷烃的选择性羟基化是一个长期存在的问题，对此没有实用的催化剂。[13]为了生产用于小烷烃选择性羟基化的基于P450 BM 3的生物催化剂，我们先前设计了这种酶以接受丙烷和乙烷（35 E11变体）。[14]尽管在体外支持的总周转率（TTN）大于5000，但该催化剂的实用性仍然有限，因为其体内性能差（见下文），这主要是由于将产物形成与辅因子消耗偶联的效率低（丙烷为17.4%，乙烷氧化为0.01%）。
Cytochrome P450 enzymes (P450s) are exceptional oxygenating catalysts [1] with enormous potential in drug discovery, chemical synthesis, bioremediation, and biotechnology.[2, 3] Compared to their natural counterparts, however, engineered P450s often exhibit poor catalytic and cofactor coupling efficiencies.[3] Obtaining native-like catalytic proficiencies is a mandatory first step towards utilizing the power of these versatile oxygenases in chemical synthesis. Cytochrome P450BM3 (119 kDa, B. megaterium) catalyzes the subterminal hydroxylation of long-chain (C12–C20) fatty acids.[4] Its high activity and catalytic self-sufficiency (heme and diflavin reductase domains are fused in a single polypeptide chain)[2, 4, 5] make P450BM3 an excellent platform for biocatalysis. However, despite numerous reports of the heme domain being engineered to accept nonnative substrates, including short-chain fatty acids, aromatic compounds, alkanes, and alkenes,[6–8] reports of preparative-scale applications of P450BM3 remain scarce.[9] P450BM3 function is finely regulated through conformational rearrangements in the heme and reductase domains and possibly also through hinged domain motions.[4, 10] Hydroxylation of fatty acids occurs almost fully coupled to cofactor (NADPH) utilization (93–96% depending on the substrate).[11] In the presence of nonnative substrates or when amino acid substitutions are introduced, the mechanisms controlling efficient catalysis in P450s are disrupted,[12] leading to the formation of reactive oxygen species and rapid enzyme inactivation.[4] High coupling efficiencies on substrates whose physicochemical properties are substantially different from the native substrates have not been achieved, and coupling efficiencies ranging from less than 1% to 30–40% are typical.[7, 8] Strategies for addressing this “coupling problem” are needed in order to take engineered P450s to larger-scale applications.Selective hydroxylation of short alkanes is a long-standing problem, for which no practical catalysts are available.[13] In an effort to produce P450BM3-based biocatalysts for selective hydroxylation of small alkanes, we previously engineered this enzyme to accept propane and ethane (35E11 variant).[14] Despite greater than 5000 total turnover (TTN) supported in vitro, the utility of this catalyst remained limited because of its poor in vivo performance (see below), which was mostly due to the low efficiencies for coupling the product formation to cofactor consumption (17.4% for propane and 0.01% for ethane oxidation).