Title | Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. |
Publication Type | Journal Article |
Year of Publication | 2010 |
Authors | de Carvalho LPedro S, Zhao H, Dickinson CE, Arango NM, Lima CD, Fischer SM, Ouerfelli O, Nathan C, Rhee K |
Journal | Chem Biol |
Volume | 17 |
Issue | 4 |
Pagination | 323-32 |
Date Published | 2010 Apr 23 |
ISSN | 1879-1301 |
Keywords | Metabolomics, 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. |
DOI | 10.1016/j.chembiol.2010.03.009 |
Alternate Journal | Chem. Biol. |
PubMed ID | 20416504 |
PubMed Central ID | PMC2878197 |
Grant List | R01 AI064768 / AI / NIAID NIH HHS / United States R01 AI064768-05 / AI / NIAID NIH HHS / United States R01 AI64768 / AI / NIAID NIH HHS / United States |
Submitted by mam2155 on March 24, 2014 - 4:19pm