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RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.

ABSTRACT: Pyridoxal 5'-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA. IMPORTANCE External stresses that disrupt metabolic components can perturb cellular functions and affect growth. A similar consequence is expected if endogenously generated metabolites are reactive and persist in the cellular environment. Here we show that the metabolic intermediate 2-aminoacrylate (2AA) causes significant cellular damage if allowed to accumulate aberrantly. Furthermore, we show that the widely conserved protein RidA prevents this accumulation by facilitating conversion of 2AA to a stable metabolite. This work demonstrates that the reactive metabolite 2AA, previously considered innocuous in the cell due to a short half-life in aqueous solution, can survive in the cellular environment long enough to cause damage. This work provides insights into the roles and persistence of reactive metabolites in vivo and shows that the RidA family of proteins is able to prevent damage caused by a reactive intermediate that is created as a consequence of PLP-dependent chemistry.

SUBMITTER: Lambrecht JA

PROVIDER: S-EPMC3565831 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.

Lambrecht Jennifer A JA Schmitz George E GE Downs Diana M DM

mBio 20130205 1

Pyridoxal 5'-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were r ...[more]

PMID: 23386433

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Project description:The Rid (YjgF/YER057c/UK114) protein superfamily was first defined by sequence homology with available protein sequences from bacteria, archaea, and eukaryotes (L. Parsons, N. Bonander, E. Eisenstein, M. Gilson, et al., Biochemistry 42:80-89, 2003, https://doi.org/10.1021/bi020541w). The archetypal subfamily, RidA (reactive intermediate deaminase A), is found in all domains of life, with the vast majority of free-living organisms carrying at least one RidA homolog. In over 2 decades, close to 100 reports have implicated Rid family members in cellular processes in prokaryotes, yeast, plants, and mammals. Functional roles have been proposed for Rid enzymes in amino acid biosynthesis, plant root development and nutrient acquisition, cellular respiration, and carcinogenesis. Despite the wealth of literature and over a dozen high-resolution structures of different RidA enzymes, their biochemical function remained elusive for decades. The function of the RidA protein was elucidated in a bacterial model system despite (i) a minimal phenotype of ridA mutants, (ii) the enzyme catalyzing a reaction believed to occur spontaneously, and (iii) confusing literature on the pleiotropic effects of RidA homologs in prokaryotes and eukaryotes. Subsequent work provided the physiological framework to support the RidA paradigm in Salmonella enterica by linking the phenotypes of mutants lacking ridA to the accumulation of the reactive metabolite 2-aminoacrylate (2AA), which damaged metabolic enzymes. Conservation of enamine/imine deaminase activity of RidA enzymes from all domains raises the likelihood that, despite the diverse phenotypes, the consequences when RidA is absent are due to accumulated 2AA (or a similar reactive enamine) and the diversity of metabolic phenotypes can be attributed to differences in metabolic network architecture. The discovery of the RidA paradigm in S. enterica laid a foundation for assessing the role of Rid enzymes in diverse organisms and contributed fundamental lessons on metabolic network evolution and diversity in microbes. This review describes the studies that defined the conserved function of RidA, the paradigm of enamine stress in S. enterica, and emerging studies that explore how this paradigm differs in other organisms. We focus primarily on the RidA subfamily, while remarking on our current understanding of the other Rid subfamilies. Finally, we describe the current status of the field and pose questions that will drive future studies on this widely conserved protein family to provide fundamental new metabolic information.

Project description:Background: Unconjugated bilirubin (UCB) is more than the final product of heme catabolism. Mildly elevated systemic bilirubin concentrations, such as in Gilbert syndrome (GS), protect against various oxidative stress-mediated and metabolic diseases, including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, cancer, and age-related disease. The Gunn rat is an animal model of hereditary hyperbilirubinemia widely used in assessing the effect of high serum bilirubin concentration in various organs. The present work aims to understand if life-long hyperbilirubinemia and bilirubin-priming might contribute to protection against atherosclerosis and diabetic nephropathy (DN) at the cellular level. Methods: Primary aortic endothelial cells and podocytes obtained from hyperbilirubinemic homozygous jj and normobilirubinemic heterozygous Nj Gunn rats were exposed to Palmitic Acid (PA) and Angiotensin II (Ang II), respectively, and the effects on cell viability and the activation of damage-related metabolic pathways evaluated. Results were validated on immortalized H5V and HK2 cells exposed to damage after UCB pretreatment. Results: In both primary cell models, cells obtained from jj Gunn rats showed as significantly higher than Nj Gunn rats at any dose of the toxic agent. Reduction in CHOP expression and IL-6 release was observed in jj primary aortic endothelial cells exposed to PA compared to Nj cells. The same occurred on H5V pretreated with Unconjugated bilirubin. Upon Ang II treatment, primary podocytes from jj Gunn rats showed lower DNA fragmentation, cleaved caspase-3, and cleaved PARP induction than primary podocytes from Nj Gunn rats. In HK2 cells, the induction by Ang II of HIF-1α and LOXl2 was significantly reduced by UCB pretreatment. Conclusion: Our data suggest that in models of atherosclerosis and DN life-long hyperbilirubinemia exposure or bilirubin-priming significantly contribute to decrease the injury by enhancing thecellular defensive response.

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RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.

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RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.

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