Affiliation:
1. Van ‘t Hoff Institute for Molecular Sciences HIMS-Biocat University of Amsterdam Science Park 904 Amsterdam 1098 XH The Netherlands
2. Institute of Biochemistry Graz University of Technology Petersgasse 12 8010 Graz Austria
Abstract
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 biocatalytic applications.