Activity-Based Metabolomic Profiling of Enzymatic Function: Identification of Rv1248c as a Mycobacterial 2-Hydroxy-3-oxoadipate Synthase Academic Article uri icon

Overview

MeSH Major

  • Metabolomics
  • Mycobacterium tuberculosis
  • 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.

publication date

  • April 23, 2010

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2878197

Digital Object Identifier (DOI)

  • 10.1016/j.chembiol.2010.03.009

PubMed ID

  • 20416504

Additional Document Info

start page

  • 323

end page

  • 32

volume

  • 17

number

  • 4