Carving the Active Site of CYP153A7 Monooxygenase for Improving Terminal Hydroxylation of Medium-Chain Fatty Acids
Graphical Make activation of C-H bond feasible : Cytochrome P450 monooxygenases (P450s) catalyze the terminal hydroxylation of fatty acids to form ω-hydroxy fatty acids. To date, available P450s for the terminal hydroxylation of fatty acids stalls at CYP153A subfamily members, which have the limitation of low activity and poor thermostability. We, therefore, adopted directed evolution to generate P450pyr mutants with higher catalytic efficiency toward medium-chain fatty acids. The results show that mutants toward C6−C12 fatty acids display improved analytical yields of ω-hydroxy fatty acids. 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 8:0 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 8:0, 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 8:0 to the high value-added product 8-hydroxyoctanoic acid.
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