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Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.

TitleActivity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.
Publication TypeJournal Article
Year of Publication2010
Authorsde Carvalho LPedro S, Zhao H, Dickinson CE, Arango NM, Lima CD, Fischer SM, Ouerfelli O, Nathan C, Rhee K
JournalChem Biol
Volume17
Issue4
Pagination323-32
Date Published2010 Apr 23
ISSN1879-1301
KeywordsMetabolomics, Mycobacterium tuberculosis, Nuclear Magnetic Resonance, Biomolecular, Oxo-Acid-Lyases
Abstract

Activity based metabolomic profiling (ABMP) allows unbiased discovery of enzymatic activities encoded by genes of unknown function, and applies liquid-chromatography mass spectrometry (LC-MS) to analyze the impact of a recombinant enzyme on the homologous cellular extract as a physiologic library of potential substrates and products. The Mycobacterium tuberculosis protein Rv1248c was incompletely characterized as a thiamine diphosphate-dependent alpha-ketoglutarate decarboxylase. Here, recombinant Rv1248c catalyzed consumption of alpha-ketoglutarate in a mycobacterial small molecule extract with matched production of 5-hydroxylevulinate (HLA) in a reaction predicted to require glyoxylate. As confirmed using pure substrates by LC-MS, (1)H-NMR, chemical trapping, and intracellular metabolite profiling, Rv1248c catalyzes C-C bond formation between the activated aldehyde of alpha-ketoglutarate and the carbonyl of glyoxylate to yield 2-hydroxy-3-oxoadipate (HOA), which decomposes to HLA. Thus, Rv1248c encodes an HOA synthase.

DOI10.1016/j.chembiol.2010.03.009
Alternate JournalChem. Biol.
PubMed ID20416504
PubMed Central IDPMC2878197
Grant ListR01 AI064768 / AI / NIAID NIH HHS / United States
R01 AI064768-05 / AI / NIAID NIH HHS / United States
R01 AI64768 / AI / NIAID NIH HHS / United States

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