期刊
CHEMISTRY & BIOLOGY
卷 17, 期 4, 页码 323-332出版社
CELL PRESS
DOI: 10.1016/j.chembiol.2010.03.009
关键词
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资金
- NIH [RO1 AI64768]
- Burroughs Wellcome Career Award in the Biomedical Sciences
- William Randolph Hearst Foundation
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 (H LA) in a reaction predicted to require glyoxylate. As confirmed using pure substrates by LC-MS, H-1-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.
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