Engineering New Substrate Specificity into the Active Site of Styrene Monooxygenases

Styrene Monooxygenases catalyze the FAD-dependent epoxidation of styrene to (S)-styrene oxide in the first step of the styrene catabolic and detoxification pathways employed by a range of microorganisms. The robust tolerance of the styrene-binding pocket to mutagenesis and the tunability of the active site chemistry of SMOs has identified these enzymes a valuable new target for development as biocatalysts with applications in chiral synthesis and bioremediation. In the present study, we systematically evaluate the impact of changing hydrophobic volume in the styrene-binding pocket of an N-terminally histidine-tagged epoxidase (NSMOA) by using the active site-directed mutants, V211A, V303A, V211I, and V303I. We find the equilibrium midpoint potential of FAD bound to these mutants to be indistinguishable from the wild-type protein, indicating that the electronic environment of the FAD is not significantly changed by the amino acid substitutions. The kinetics of the epoxidation reaction of a library of subst...