Kinetic mechanism of dimethylsulfone monooxygenase and its immobilized magnetic nanoparticles

Abstract

The dimethyl sulfone monooxygenase system is a two-component flavoprotein catalyzing the monooxygenation of dimethyl sulfone (DMSO2) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor catalyzing flavin reduction using NADH. The oxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH– with a Kd of 17.4 ± 0.9 µM and 4.08 ± 0.8 mM, respectively. The binding of FMN to DMSMO is required prior to binding DMSO2. Substituting reduced DMSR with FMNH– demonstrated the same oxidation kinetics, indicating that FMNH- from DMSR was transferred to DMSMO. The oxidation of FMNH–:DMSMO, with and without DMSO2 did not generate any flavin adducts for monooxygenation. Therefore, H2O2 is likely to be the reactive agent to attack the substrate. The H2O2 assay results demonstrated production of H2O2 from the oxidation of FMNH–:DMSMO, whereas H2O2 was not detected in the presence of DMSO2, confirming H2O2 utilization. The rate constant for methanesulfinate formation determined from rapid-quenched flow and the rate constant for flavin oxidation were similar, indicating that H2O2 rapidly reacts with dimethyl sulfone. Both enzymes have been co-immobilized onto Ni-NTA functionalized magnetic nanoparticles (CoFe2O4@SiO2–Ni/NTA) to improve enzyme stability in different environments and enable the enzymes to be recovered for reuse.