The Response to 2-Aminoacrylate Differs in Escherichia coli and Salmonella enterica, despite Shared Metabolic Components

Abstract

The metabolic network of an organism includes the sum total of the biochemical reactions present. In microbes, this network has an impeccable ability to sense and respond to perturbations caused by internal or external stimuli. The metabolic potential (i.e., network structure) of an organism is often drawn from the genome sequence, based on the presence of enzymes deemed to indicate specific pathways. Escherichia coli and Salmonella enterica are members of the Enterobacteriaceae family of Gram-negative bacteria that share the majority of their metabolic components and regulatory machinery as the "core genome." In S. enterica, the ability of the enamine intermediate 2-aminoacrylate (2AA) to inactivate a number of pyridoxal 5=-phosphate (PLP)-dependent enzymes has been established in vivo. In this study, 2AA metabolism and the consequences of its accumulation were investigated in E. coli. The data showed that despite the conservation of all relevant enzymes, S. enterica and E. coli differed in both the generation and detrimental consequences of 2AA. In total, these findings suggest that the structure of the metabolic network surrounding the generation and response to endogenous 2AA stress differs between S. enterica and E. coli. IMPORTANCE This work compared the metabolic networks surrounding the endogenous stressor 2-aminoacrylate in two closely related members of the Enterobacteriaceae. The data showed that despite the conservation of all relevant enzymes in this metabolic node, the two closely related organisms diverged in their metabolic network structures. This work highlights how a set of conserved components can generate distinct network architectures and how this can impact the physiology of an organism. This work defines a model to expand our understanding of the 2-aminoacrylate stress response and the differences in metabolic structures and cellular milieus between S. enterica and E. coli. KEYWORDS 2-aminoacrylate, PLP-dependent enzymes, RidA, metabolic network structure T he metabolic networks of microbes are characterized by robustness and redundancy that ensure their ability to absorb perturbations caused by internal and external stress (1-4). Our ability to predict the metabolic architecture of an organism and how it will respond to perturbations is poorly developed. Despite the fact that metabolic components encoded by the genome can be defined, it is not yet clear how accurately the metabolic capacity and robustness of an organism are predicted by the mere presence of these metabolic components. In some cases, the status of central and linear pathways that are predicted from genome annotation can be tested by simple growth phenotypes. For instance, the lack of a single histidine biosynthetic enzyme (i.e., ATP phosphoribosyltransferase [HisG]; EC 2.4.2.17) predicts that there will be a requirement for exogenous histidine. In contrast, growth phenotypes resulting from differen-

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa

“…The third Rid protein, STM1549, lacks an active site Arg residue and does not deaminate imines. E. coli K-12 encodes three Rid proteins, including two RidA homologs that have 2AA deaminase activity: RidA and TdcF (8). Assessing the RidA paradigm in numerous organisms highlighted conserved features and uncovered distinct properties that reflect the consequence of specific metabolic architectures.…”

“…Assessing the RidA paradigm in numerous organisms highlighted conserved features and uncovered distinct properties that reflect the consequence of specific metabolic architectures. A ridA mutation in S. enterica causes a number of growth defects, while growth is unaffected in an E. coli double mutant (ridA tdcF) unless 2AA accumulation is artificially increased (8). In yeast, inactivating the mitochondrial RidA homolog (Mmf1) results in significant growth and biochemical defects, while the loss of the cytoplasmic homolog (Hmf1) fails to have detectable consequences (9)(10)(11).…”

Integrated Metabolomics and Transcriptomics Suggest the Global Metabolic Response to 2-Aminoacrylate Stress in Salmonella enterica

“…A majority of the metabolites (10/23) were related to, or the product of, amino acid biosynthetic pathways and a significant number (5/23) were components nucleotide metabolism. The former was expected based on the prevalence of PLP-dependent enzymes (targets of 2AA damage) in amino acid metabolism [22][23][24][25]31], while a direct role of PLP-dependent enzymes in nucleotide metabolism was not obvious. The 37 metabolites that could be confidently identified represented a small sample from all metabolites produced by S. enterica and thus limited the conclusions that could be made about the global metabolic state of S. enterica ridA mutants.…”

“…For example, IlvE, AspC, and aromatic amino acid aminotransferase (TyrB; EC: 2.6.1.57) facilitate the synthesis of phenylalanine from phenylpyruvate [42]. Damage of IlvE [21] and AspC [24] by 2AA may explain the phenylalanine decrease in a ridA background. Similarly, phosphoserine transaminase (SerC, EC: 2.6.1.52) facilitates the conversion of 3-phosphooxypyruvate to O-phospho-L-serine during serine biosynthesis, while SerC and N-succinyldiaminopimelate aminotransferase (ArgD, EC: 2.6.1.17) catalyze N-succinyldiaminopimelate production from N-succinyl-L-amino-6-ketopimelate during lysine biosynthesis (Figure 3) [41,43].…”

“…In the absence of RidA, the enamine 2-aminoacrylate (2AA), a well-characterized inhibitor of multiple pyridoxal 5′-phosphate (PLP)-dependent enzymes, accumulates ( Figure 1). Accumulated 2AA inhibits serine hydroxymethyltransferase (GlyA, EC: 2.1.2.1), aspartate aminotransferase (AspC; EC: 2.6.1.1), alanine racemase (Alr and DadX; EC: 5.1.1.1), and branched-chain amino acid aminotransferase (IlvE; EC: 2.6.1.42) [21][22][23][24], reducing their total cellular activity by 20%-60%. Figure 1.…”