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

Abstract

SUMMARY Activity based metabolomic profiling (ABMP) allows unbiased discovery of enzymatic activities encoded by genes of unknown function. ABMP 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 α-ketoglutarate decarboxylase. Here, recombinant Rv1248c catalyzed consumption of α–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, 1H-NMR, chemical trapping, and intracellular metabolite profiling, Rv1248c catalyzes C-C bond formation between the activated aldehyde of α–ketoglutarate and the carbonyl of glyoxylate to yield 2-hydroxy-3-oxoadipate (HOA), which decomposes to HLA. Thus, Rv1248c encodes a HOA synthase (HOAS).

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

“…S9). LC-MS analysis of the metabolome of wild-type, Δrv1692, and complemented strains was performed as previously described (8,25). Targeted metabolomic analysis shows an increase of the pool size of glycerol phosphate in the Δrv1692 compared with the wild-type (Fig.…”

“…In this method, we prepared an extract of polar metabolites from mycobacteria (17) and used it as a library of potential in vitro substrates. This small-molecule extract (SME) contains ∼1,700 unique ions (8). The identity and the abundance of ionizable polar small molecules in the SME are determined in both the absence and the presence of the purified Rv1692 and are analyzed in both positive and negative ion modes.…”

“…This observation, together with the presence of an extra domain and a differentially evolved cap domain in Rv1692, suggest that Rv1692 has a distinct substrate specificity and function. To search for a substrate of Rv1692, we applied ABMP (8). In this method, we prepared an extract of polar metabolites from mycobacteria (17) and used it as a library of potential in vitro substrates.…”

“…ABMP employs liquid chromatography coupled with accurate time-of-flight mass spectrometry to separate, identify, and quantify potential substrates and products in a mycobacterial extract of small molecules acting as a representative source of physiologically relevant substrates, cofactors, and allosteric regulators. One of the main advantages of ABMP is the lack of any inherent bias toward the number and type of substrates or toward the nature of a catalytic mechanism (8).…”

“…SME was prepared as described earlier (8). Briefly, the Mycobacterium bovis bacillus Calmette-Guérin pellet was suspended in 10 mL acidic ACN solution (acetonitrile, 0.2% acetic acid), and cells were disrupted by sonication.…”