Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.
Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.
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DOI：
10.1016/j.chembiol.2010.03.009
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发表时间：
2010-04-23
影响因子：
--
通讯作者：
Rhee KY
中科院分区：
文献类型：
--
作者：
de Carvalho LP;Zhao H;Dickinson CE;Arango NM;Lima CD;Fischer SM;Ouerfelli O;Nathan C;Rhee KY
基于活性的代谢组学分析（ABMP）允许无偏地发现由未知功能的基因编码的酶活性。ABMP应用液相色谱-质谱（LC-MS）分析重组酶对同源细胞提取物的影响，作为潜在底物和产物的生理文库。结核分枝杆菌蛋白Rv 1248 c是一种二磷酸硫胺素依赖的α-酮戊二酸脱羧酶。在这里，重组Rv 1248 c催化消耗分枝杆菌小分子提取物中的α-酮戊二酸，并在预测需要乙醛酸的反应中产生匹配的5-羟基乙酰丙酸（HLA）。通过LC-MS、1H-NMR、化学捕获和细胞内代谢产物分析使用纯底物证实，Rv 1248 c催化α-酮戊二酸的活化醛与乙醛酸的羰基之间的C-C键形成，生成2-羟基-3-氧代己二酸（HOA），其分解为HLA。因此，Rv 1248 c编码HOA合酶（HOAS）。
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).
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