Journal
ORGANIC & BIOMOLECULAR CHEMISTRY
Volume 19, Issue 15, Pages 3441-3450Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/d1ob00015b
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Funding
- European Union [764920]
- Marie Curie Actions (MSCA) [764920] Funding Source: Marie Curie Actions (MSCA)
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This study demonstrates the independent functionality of the oxygenase in two-component flavoprotein monooxygenases and the mechanism of FMNH2 transport by free diffusion. It also highlights the importance of balancing FMN reduction and enzymatic (re)oxidation in a reductase-free system.
Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.
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