Biochemical Characterization of Thermocrispum agreste TheA: A Flavin-Dependent N-hydroxylating Enzyme
Mena Aguilar, Didier Philippe
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N-hydroxylating monooxygenases (NMOs) are Class B flavin-dependent monooxygenases found only in fungi and bacteria. These enzymes catalyze the hydroxylation of nucleophilic primary amines, such as those found in histamine, L-ornithine, L-lysine, and small aliphatic diamines. The hydroxamate moiety produced by this reaction is key for the production of siderophores, small chelating compounds that allow survival in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. Herein we present the characterization of TheA, an NMO from Thermocrispum agreste. The enzyme mechanism was studied using steady state kinetic measurements, thermostability, and stopped flow spectrophotometry assays. Using these techniques, the catalytic rates, substrate binding affinities, thermal stability, and coenzyme specificities were determined. Additionally, NADPH analogues were produced to use as tools to study FAD reduction in NMOs. An unspecific reduction reaction of NADP+ using NaB2H4 yielded [6-2H]-NADPH, [2-2H]-NADPH, and [4-2H]-NADPH. Compound identity was confirmed by mass spectrometry and unidimensional proton nuclear magnetic resonance (NMR). Results presented in this thesis lay the foundation for future studies of NMOs using NADPH analogues. In conjunction, these results will improve the general knowledge and understanding of flavoenzymes, ornithine monooxygenases, and their associated mechanisms.
- Masters Theses