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Abstract Flavin‐dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer–Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented by a bioinformatics analysis, that clarify the early stages of their catalytic cycle. We have elucidated the intricate binding mechanism of NADPH and L‐Orn to a class B monooxygenase, the ornithine hydroxylase from known as SidA. Our investigation involved a comprehensive characterization of the conformational changes associated with the FAD (Flavin Adenine Dinucleotide) cofactor, transitioning from theoutto theinposition. Furthermore, we explored the rotational dynamics of the nicotinamide ring of NADPH, shedding light on its role in facilitating FAD reduction, supported by experimental evidence. Finally, we also analyzed the extent of conservation of two Tyr‐loops that play critical roles in the process. 

CreE is a flavin‐dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L‐aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group in which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L‐aspartate, aiding in future enzyme engineering efforts. Steady‐state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L‐aspartate. Analysis of the rapid‐reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a‐hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site‐directed mutagenesis implicated T65, R291, and R440 in the binding L‐aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.