Carving the Active Site of CYP153A7 Monooxygenase for Improving Terminal Hydroxylation of Medium-Chain Fatty Acids.

ABSTRACT

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-C80 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 (C80, 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 C80 to the high value-added product 8-hydroxyoctanoic acid.