Methods for Biochemical Characterization of Flavin-Dependent N-Monooxygenases Involved in Siderophore Biosynthesis

Abstract

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.

Department(s)

Chemistry

Keywords and Phrases

Enzyme assays; Flavin-dependent monooxygenases (FMOs); Fluorescence anisotropy; Iodine-oxidation; Kinetic isotope effects; N-hydroxylating monooxygenases (NMOs); Oxygraphy; Siderophores

International Standard Book Number (ISBN)

978-044329678-9

International Standard Serial Number (ISSN)

1557-7988; 0076-6879

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

01 Jan 2024

PubMed ID

39155115

Link to Full Text

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