Rational design of styrene monooxygenase mutants with altered substrate preference

Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene derivatives with an α- or β-substituent. Based on the X-ray crystal structure of the oxygenase subunit of styrene monooxygenase, molecular docking of α-ethylstyrene was performed to identify adjacent residues. Four amino acid substitutions (R43A, L44A, L45A, and N46A) were introduced into the enzyme by site-directed mutagenesis. All four mutations led to a change of substrate preference. The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate α-ethylstyrene.