Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO – a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH₂ from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45–50 Å) facilitated by the flexible linker‘s loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (k_red=110 s⁻¹ for bound FAD_ox), identifying free FAD_ox binding as the rate-determining step. The aerobic oxidation of FADH₂ (k_ox=90 s⁻¹) and subsequent decomposition to FAD_ox and H₂O₂ demonstrated StyA′s protective effect on the bound hydroperoxoflavin (k^dec=0.2 s⁻¹) compared to free cofactor (k_dec=1.8 s⁻¹). At varied styrene concentrations, k_ox for FADH₂ ranged from 80 to 120 s⁻¹. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO′s ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH₂ channelling between subunits. This work contributes to comprehending FADH₂ transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.