Kinetic Characterization and Identification of Key Active Site Residues of the L‐Aspartate N‐Hydroxylase, CreE

• Published in: Chembiochem : a European journal of chemical biology Vol. 25; no. 14; pp. e202400350 - n/a
• Main Authors: Johnson, Sydney B., Valentino, Hannah, Sobrado, Pablo
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 15.07.2024
• Subjects: Binding; Biosynthesis; Chemical reactions; Dehydration; Flavin; Flavin-Dependent Monooxygenase; Functional groups; Kinetics; Mutagenesis; Natural Products; Nicotinamide adenine dinucleotide; Oxidation; Oxygenation; Reaction kinetics; Residues; Structural analysis; flavin monooxygenase, cremeomycin, nitrosuccinate, C4a-hydroperoxyflavin, N-monooxygenase
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202400350

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 for 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. This work characterizes the catalytic mechanism of the multiple oxidizing flavin‐dependent monooxygenase, CreE, using steady‐state and rapid‐reaction kinetic approaches. Analysis of the active site revealed residues that are involved in binding L‐aspartate and are conserved in related enzymes, providing the crucial foundation for enzyme engineering efforts for biomedical and synthetic applications.