Dennig Alexander, Weingartner Alexandra Maria, Kardashliev Tsvetan, Müller Christina Andrea, Tassano Erika, Schürmann Martin, Ruff Anna Joëlle, Schwaneberg Ulrich
Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010, Graz, Austria.
Chemistry. 2017 Dec 19;23(71):17981-17991. doi: 10.1002/chem.201703647. Epub 2017 Nov 23.
Aromatic hydroxylation of pseudocumene (1 a) and mesitylene (1 b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1 b (94 %), whereas 1 a was hydroxylated to a large extent on benzylic positions (46-64 %). Site-saturation mutagenesis generated a new P450 BM3 mutant, herein named "variant M3" (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3- to 8-fold) and activity (75- to 230-fold) for the conversion of 1 a and 1 b. Additional π-π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also selectivity toward aromatic hydroxylation of 1 a (61 to 75 %). Under continuous nicotinamide adenine dinucleotide phosphate recycling, the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor trimethylhydroquinone (3 a; 35 % selectivity; 0.18 mg mL ) directly from 1 a. In the case of 1 b, overoxidation leads to dearomatization and the formation of a valuable p-quinol synthon that can directly serve as an educt for the synthesis of 3 a. Detailed product pattern analysis, substrate docking, and mechanistic considerations support the hypothesis that 1 a binds in an inverted orientation in the active site of P450 BM3 WT, relative to P450 BM3 variant M3, to allow this change in chemoselectivity. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O .
用细胞色素P450 BM3对均三甲苯（1 a）和偏三甲苯（1 b）进行芳香族羟基化反应，可生成用于合成α-生育酚的关键酚类结构单元。细胞色素P450 BM3野生型（WT）催化1 b的选择性芳香族羟基化反应（94 %），而1 a在苄基位置上有很大程度的羟基化（46 - 64 %）。位点饱和诱变产生了一种新的细胞色素P450 BM3突变体，在此命名为“变体M3”（R47S、Y51W、A330F、I401M），对于1 a和1 b的转化，其偶联效率显著提高（3至8倍），活性提高（75至230倍）。由A330F突变引入的额外π-π相互作用不仅提高了产率和偶联效率，还提高了对1 a芳香族羟基化反应的选择性（从61 %提高到75 %）。在烟酰胺腺嘌呤二核苷酸磷酸持续循环的条件下，新型细胞色素P450 BM3变体M3能够直接从1 a生成关键的生育酚前体三甲基对苯二酚（3 a；选择性为35 %；0.18 mg mL ）。对于1 b，过氧化会导致去芳香化并形成一种有价值的对苯二酚合成子，该合成子可直接用作合成3 a的原料。详细的产物模式分析、底物对接和机理研究支持了这样一种假设：相对于细胞色素P450 BM3变体M3，1 a在细胞色素P450 BM3 WT的活性位点中以反向取向结合，从而导致这种化学选择性的变化。本研究提供了一条合成α-生育酚关键酚类合成子的酶促路线，并首次对用O 对三甲苯进行直接芳香族羟基化反应和去芳香化反应的催化作用及机理进行了深入研究。
