Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase
Tanja Knaus1, Peter Macheroux2, Francesco G Mutti1
1Van 't Hoff Institute for Molecular Sciences, HIMS-Biocat, University of Amsterdam, Science Park 904, Amsterdam, 1098 XH, The Netherlands.
Chembiochem : a European Journal of Chemical Biology
|February 2, 2024
Summary
This study engineered a fused styrene monooxygenase (Fus-SMO) enzyme, revealing enhanced FADH2 channeling and improved catalytic efficiency. Fus-SMO demonstrates superior performance compared to natural systems, advancing biocatalysis.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Styrene monooxygenase (SMO) is a two-component enzyme system crucial for styrene epoxidation.
- The mechanism of FADH2 transfer between SMO components (StyA and StyB) is debated, with possibilities including diffusion or channeling.
Purpose of the Study:
- To characterize a novel chimera enzyme, Fus-SMO, created by fusing StyA and StyB with a flexible linker.
- To investigate the FADH2 transfer mechanism and catalytic efficiency of Fus-SMO.
- To explore protein fusion as a strategy for enhancing biocatalytic performance.
Main Methods:
- Protein expression and purification of Fus-SMO.
- In silico modeling to predict enzyme structure and subunit arrangement.
- Pre-steady-state kinetic analysis to determine reaction rates and mechanisms.
- Spectroscopic studies to monitor cofactor redox states and product formation.
- Enzyme activity assays measuring NADH consumption and styrene epoxidation.
Main Results:
- Fus-SMO forms a trimer with a single bound FAD molecule.
- In silico analysis suggests a 45-50 Å distance between subunits mediated by a flexible linker.
- Kinetic studies revealed FADH2 reduction (kred=110 s⁻¹) and aerobic oxidation (kox=90 s⁻¹).
- StyA demonstrated a protective effect on bound hydroperoxoflavin, reducing its decomposition rate.
- Fus-SMO achieved quantitative coupling efficiency, surpassing natural SMOs and indicating enhanced FADH2 channeling.
Conclusions:
- Protein fusion via a flexible linker enhances FADH2 channeling in SMO.
- Fus-SMO exhibits significantly improved catalytic efficiency for styrene epoxidation.
- This work provides insights into FADH2 transfer mechanisms and highlights the potential of protein engineering for biocatalysis.
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