title: Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.
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.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA.
authors.forename: Luiz Pedro S
authors.initials: LP
authors.lastname: de Carvalho
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Caitlyn E
authors.initials: CE
authors.lastname: Dickinson
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Arango
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Lima
authors.forename: Steven M
authors.initials: SM
authors.lastname: Fischer
authors.forename: Ouathek
authors.initials: O
authors.lastname: Ouerfelli
authors.forename: Carl
authors.initials: C
authors.lastname: Nathan
authors.forename: Kyu Y
authors.initials: KY
authors.lastname: Rhee
mesh_terms: D019880:Aldehyde-Ketone Transferases; D055432:Metabolomics / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D007652:Oxo-Acid-Lyases / Q000378:metabolism*
chemical_list: D019880:Aldehyde-Ketone Transferases; C021414:2-hydroxy-3-oxoadipate synthase; D007652:Oxo-Acid-Lyases