Masters Thesis

Mechanistic studies of StyA2B a self-sufficient styrene monooxygenase from R. opacus 1CP.

Styrene Monooxygenases (SMOs) catalyze the epoxidation reaction of styrene with high enantioselectivity, which establishes them as potential biocatalysts in chemical synthesis and bioremediation processes. In this work, we conducted a mechanistic study on StyA2B, a naturally fused SMO from R. opacus 1CP. The kinetic studies performed revealed that the reductase domain of StyA2B catalyzes the reduction of FAD in a sequential ternary mechanism. Steady-state kinetic studies of the reaction of StyA2B with NADH and styrene showed that styrene binds weakly at low concentrations, but the epoxidase shows strong positive cooperativity between its binding ligands. No substrate inhibition was observed at high styrene concentrations. The addition of the partner epoxidase, StyAl, from the StyAl/StyA2B system binds styrene non-cooperatively, but with higher affinity and StyAl was observed to increase the catalytic activity of the StyA2B reductase. In contrast, StyAl had no detected effect on the kinetic mechanism of SMOB, a flavin reductase native to P. putida. This suggests that a specific protein-protein interaction may occur in the StyAl/StyA2B system. The equilibrium midpoint potential of FAD to StyA2B was measured to obtain information about the electronic environment of the FAD bound to StyA2B. A significant shift of the bound-FAD midpoint potential to a value more positive than that of free FAD was detected. This indicates that the reduced FAD binds more tightly than oxidized FAD to StyA2B. This observation is congruent with the steady-state ternary complex mechanism, which involves the binding of oxidized FAD and dissociation and transport of reduced FAD from the reductase to the epoxidase. The goal of the thesis is to provide clear description of kinetic behavior of the StyAl/Sty A2B system. This will yield insight into unique features of this two-component styrene monooxygenase and may help to better establish its utility as a valuable biocatalyst.

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