Methods for biochemical characterization of flavin-dependent N-monooxygenases involved in siderophore biosynthesis.

Siderophores are essential molecules released by some bacteria and fungi in iron-limiting environments to sequester ferric iron, satisfying metabolic needs. Flavin-dependent N-hydroxylating monooxygenases (NMOs) catalyze the hydroxylation of nitrogen atoms to generate important siderophore functional groups such as hydroxamates. It has been demonstrated that the function of NMOs is essential for virulence, implicating these enzymes as potential drug targets. This chapter aims to serve as a resource for the characterization of NMO's enzymatic activities using several biochemical techniques. We describe assays that allow for the determination of steady-state kinetic parameters, detection of hydroxylated amine products, measurement of the rate-limiting step(s), and the application toward drug discovery efforts. While not exhaustive, this chapter will provide a foundation for the characterization of enzymes involved in siderophore biosynthesis, allowing for gaps in knowledge within the field to be addressed.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

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

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.

Kinetic characterization of a flavin-dependent monooxygenase from the insect food crop pest, Zonocerus variegatus.

Zonocerus variegatus, or the painted grasshopper, is a food crop pest endemic in Western and Central Africa. Agricultural industries in these regions rely heavily on natural defense mechanisms to control the grasshopper population such as plant-secreted alkaloid compounds. In recent years, the Z. variegatus population has continued to rise due to acquired resistance to alkaloids. Here we focus on the kinetic characterization of a flavin-dependent monooxygenase, ZvFMO, that catalyzes the nitrogen oxidation of many of these alkaloid compounds and confers resistance to the insect. Expression and purification of ZvFMO through a traditional E. coli expression system was successful and provided a unique opportunity to characterize the catalytic properties of an FMO from insects. ZvFMO was found to catalyze oxidation reactions of tertiary nitrogen atoms and the sulfur of cysteamine. Using stopped-flow spectroscopy, we have determined the kinetic mechanism of ZvFMO. We assessed F383 for its involvement in substrate binding, which was previously proposed, and determined that this residue does not play a major role in binding substrates. Through molecular docking, we identified N304 and demonstrated that this residue plays a role in substrate binding. The role of K215 was studied and was shown that it plays a critical role in NAD(P)H binding and cofactor selectivity.