Significance of active site volume mutations in redefining the catalytic specificity of styrene monooxygenase

Styrene Monooxygenases (SMO) from Pseudomonas putida (S12) is a two-component flavoenzyme system that catalyzes the FAD- and NADH-dependent epoxidation of styrene to (S)-styrene oxide in the first step of the styrene catabolic and detoxification pathways. 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 component of styrene monooxygenase, NSMOA, through active site-directed mutagenesis. 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. Single turnover kinetic experiments indicate that the substrate specificity of NSMOA (V303A) shifts to favor epoxidation of sterically-bulky substrates with substitution in the para-position of styrene. 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.