Borchert Andrew J, Gouveia Goncalo J, Edison Arthur S, Downs Diana M
Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Department of Biochemistry, University of Georgia, Athens, Georgia, USA.
mSystems. 2020 Mar 10;5(2):e00843-19. doi: 10.1128/mSystems.00843-19.
The reactive intermediate deaminase RidA (EC 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5'-phosphate (PLP)-dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted proton nuclear magnetic resonance (H NMR) metabolomics to determine whether the metabolic state of an mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the metabolic differences for a mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled. The accumulation of the reactive enamine intermediate 2-aminoacrylate (2AA) elicits global metabolic stress in many prokaryotes and eukaryotes by simultaneously damaging multiple pyridoxal 5'-phosphate (PLP)-dependent enzymes. This work employed H NMR to expand our understanding of the consequence(s) of 2AA stress on metabolite pools and effectively identify the metabolic changes stemming from one damaged target: GlyA. This study shows that nutrient supplementation during H NMR metabolomics experiments can disentangle complex metabolic outcomes stemming from a general metabolic stress. Metabolomics shows great potential to complement classical reductionist approaches to cost-effectively accelerate the rate of progress in expanding our global understanding of metabolic network structure and physiology. To that end, this work demonstrates the utility in implementing nutrient supplementation and genetic perturbation into metabolomics workflows as a means to connect metabolic outputs to physiological phenomena and establish causal relationships.
反应性中间物脱氨酶RidA（EC 3.5.99.10）在所有生命域中都保守，并使反应性烯胺物种脱氨。当突变体在基本培养基中生长时，2-氨基丙烯酸酯（2AA）会积累，损害几种依赖磷酸吡哆醛（PLP）的酶，并引发明显的生长缺陷。遗传学研究表明，丝氨酸羟甲基转移酶（GlyA）受损以及由此导致的5,10-亚甲基四氢叶酸（5,10-mTHF）消耗是观察到的生长缺陷的原因。然而，2AA对GlyA造成损伤后的下游代谢后果仍相对未被探索。本研究旨在使用非靶向质子核磁共振（H NMR）代谢组学来确定通过表征生长表型是否能准确反映突变体的代谢状态。数据支持了以下结论：突变体中的代谢变化是由于IlvA依赖性产生2AA所致，并且这些变化中的大多数是GlyA受损的结果。虽然突变体的许多代谢差异可以得到解释，但某些代谢物的变化不易建模，这表明仍有额外水平的代谢复杂性有待揭示。反应性烯胺中间体2-氨基丙烯酸酯（2AA）的积累通过同时损害多种依赖磷酸吡哆醛（PLP）的酶，在许多原核生物和真核生物中引发全局代谢应激。这项工作采用H NMR来扩展我们对2AA应激对代谢物库的影响的理解，并有效地识别源于一个受损靶点GlyA的代谢变化。这项研究表明，在H NMR代谢组学实验期间补充营养可以解开由一般代谢应激引起的复杂代谢结果。代谢组学显示出巨大的潜力，可以补充经典的还原论方法，以经济高效地加快我们对代谢网络结构和生理学的全球理解的进展速度。为此，这项工作证明了在代谢组学工作流程中实施营养补充和基因扰动作为将代谢输出与生理现象联系起来并建立因果关系的一种手段的实用性。
