Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency.
Chembiochem
; 25(7): e202300833, 2024 Apr 02.
Article
em En
| MEDLINE
| ID: mdl-38306174
ABSTRACT
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 (45-50â
Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (kred=110â
s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox=90â
s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec=0.2â
s-1) compared to free cofactor (kdec=1.8â
s-1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120â
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.
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MEDLINE
Assunto principal:
Oxigenases
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Peróxido de Hidrogênio
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article