Styrene monooxygenases (SMOs) occur at the entry point for styrene into microbial catabolism where they catalyze the NADH-dependent, enantioselective synthesis of (S)-styrene oxide. There is significant interest in elucidating the mechanism of these enzymes and their development into biocatalysts for green organic chemical synthesis and environmental remediation. The present work focuses on StyA1, the monooxygenase of the two-component flavoprotein, StyA1/StyA2B isolated from Rhodococcus opacus. We have used a combination of steady state and pre-steady state kinetic studies of the reaction of StyA1 with styrene and styrene analogs to evaluate the epoxidation mechanism of this enzyme. We find that styrene interacts with StyA1 with an affinity 10-20 times lower than that observed for the related Pseudomonas enzyme, NSMOA. The kinetics of styrene epoxidation by StyA1 is about 15-times slower than that of NSMOA whereas the hydroxyl-FAD dehydration kinetics are similar for both enzymes. StyA1 has a unique substrate specificity favoring 4-vinyl anisole and 3-vinyl pyridine, over styrene. The epoxidation of a-methyl styrene ( k = 8.4 s-1 ) is nearly 3-times faster than the styrene epoxidation reaction. The epoxidation rate constant of StyA1 increases as a function of decreasing pH with an apparent pKa &lt; 4 and is not affected by chloride ions up to 250 mM. This is quite different from the epoxidation reaction of NSMOA, which is sensitive to anions and controlled by a pKa ~ 7.7. Elucidation of the structural and mechanistic features distinguishing the kinetic behavior of these related enzymes will be helpful in their development as biocatalysts.

Mechanistic studies of StyA1 styrene monooxygenase from Rhodococcus Opacus

253rd National Meeting (2017)

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