Huang Wei-Cheng, Westlake Andrew C G, Maréchal Jean-Didier, Joyce M Gordon, Moody Peter C E, Roberts Gordon C K
Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK.
J Mol Biol. 2007 Oct 26;373(3):633-51. doi: 10.1016/j.jmb.2007.08.015. Epub 2007 Aug 21.
Cytochrome P450BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase, is a member of a very large superfamily of monooxygenase enzymes. The available crystal structures of the enzyme show non-productive binding of substrates with their omega-end distant from the iron in a hydrophobic pocket at one side of the active site. We have constructed and characterised mutants in which this pocket is filled by large hydrophobic side-chains replacing alanine at position 82. The mutants having phenylalanine or tryptophan at this position have very much (approximately 800-fold) greater affinity for substrate, with a greater conversion of the haem iron to the high-spin state, and similarly increased catalytic efficiency. The enzyme as isolated contains bound palmitate, reflecting this much higher affinity. We have determined the crystal structure of the haem domain of the Ala82Phe mutant with bound palmitate; this shows that the substrate is binding differently from the wild-type enzyme but still distant from the haem iron. Detailed analysis of the structure indicates that the tighter binding in the mutant reflects a shift in the conformational equilibrium of the substrate-free enzyme towards the conformation seen in the substrate complex rather than differences in the enzyme-substrate interactions. On this basis, we outline a sequence of events for the initial stages of the catalytic cycle. The Ala82Phe and Ala82Trp mutants are also very much more effective catalysts of indole hydroxylation than the wild-type enzyme, suggesting that they will be valuable starting points for the design of mutants to catalyse synthetically useful hydroxylation reactions.
来自巨大芽孢杆菌的细胞色素P450BM3（CYP102A1）是一种脂肪酸羟化酶，属于非常大的单加氧酶超家族成员。该酶已有的晶体结构显示，底物在活性位点一侧的疏水口袋中与铁的ω端距离较远，呈非生产性结合。我们构建并表征了一些突变体，其中该口袋被大的疏水侧链填充，取代了82位的丙氨酸。在该位置具有苯丙氨酸或色氨酸的突变体对底物的亲和力大大提高（约800倍），血红素铁向高自旋态的转化率更高，催化效率也同样提高。分离得到的酶含有结合的棕榈酸酯，这反映了其更高的亲和力。我们测定了结合棕榈酸酯的Ala82Phe突变体血红素结构域的晶体结构；结果表明底物的结合方式与野生型酶不同，但仍远离血红素铁。对结构的详细分析表明，突变体中更强的结合反映了无底物酶的构象平衡向底物复合物中所见构象的转变，而不是酶-底物相互作用的差异。在此基础上，我们概述了催化循环初始阶段的一系列事件。Ala82Phe和Ala82Trp突变体也是比野生型酶更有效的吲哚羟化催化剂，这表明它们将是设计催化合成有用羟化反应的突变体的有价值的起点。
