Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.
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.
de Carvalho
Luiz Pedro S
LP
Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA.
Zhao
Hong
H
Dickinson
Caitlyn E
CE
Arango
Nancy M
NM
Lima
Christopher D
CD
Fischer
Steven M
SM
Ouerfelli
Ouathek
O
Nathan
Carl
C
Rhee
Kyu Y
KY
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*
D019880:Aldehyde-Ketone Transferases; C021414:2-hydroxy-3-oxoadipate synthase; D007652:Oxo-Acid-Lyases