State Key Laboratory of Bioreactor Engineering and, Shanghai Collaborative Innovation Centre for Biomanufacturing and, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai, 200237, China.
Hubei Collaborative Innovation Center for Green Transformation of Bio-resources Hubei Key Laboratory of Industrial Biotechnology, College of Life Science, Hubei University, Wuhan, 430062, China.
Chembiochem. 2022 May 4;23(9):e202200063. doi: 10.1002/cbic.202200063. Epub 2022 Mar 23.
The P450-mediated terminal hydroxylation of non-activated C-H bonds is a chemically challenging reaction. CYP153A7 monooxygenase, discovered in Sphingomonas sp. HXN200, belongs to the CYP153A subfamily and shows a pronounced terminal selectivity. Herein, we report the significantly improved terminal hydroxylation activity of CYP153A7 by redesign of the substrate binding pocket based on molecular docking of CYP153A7-C and sequence alignments. Some of the resultant single mutants were advantageous over the wild-type enzyme with higher reaction rates, achieving a complete conversion of n-octanoic acid (C 1 mM) in a shorter time period. Especially, a single-mutation variant, D258E, showed 3.8-fold higher catalytic efficiency than the wild type toward the terminal hydroxylation of medium-chain fatty acid C to the high value-added product 8-hydroxyoctanoic acid.
P450 介导的非活化 C-H 键末端羟化是一种具有挑战性的化学反应。在 Sphingomonas sp. HXN200 中发现的 CYP153A7 单加氧酶属于 CYP153A 亚家族，表现出明显的末端选择性。在此，我们通过基于 CYP153A7-C 的分子对接和序列比对，对底物结合口袋进行重新设计，显著提高了 CYP153A7 的末端羟化活性。一些所得的单点突变体比野生型酶具有更高的反应速率，在更短的时间内实现了 n-辛酸（C 1mM）的完全转化。特别是，单点突变变体 D258E 对中链脂肪酸 C 的末端羟化表现出比野生型高 3.8 倍的催化效率，生成高附加值产物 8-羟基辛酸。
