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

Chembiochem. 2024 Feb 2:e202300833. doi: 10.1002/cbic.202300833. Online ahead of print.ABSTRACTThe 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 (kred=110 s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox= 90 s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec= 0.2 s-1) compared to free cofactor (kdec= 1.8 s-1). At varied styrene concentrations, kox 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 bioca...

Source: Chembiochem - February 2, 2024 Category: Biochemistry Authors: Tanja Knaus Peter Macheroux Francesco Mutti Source Type: research