Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.

Endogenous synthesis of 2-aminoacrylate contributes to cysteine sensitivity in Salmonella enterica.

RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5'-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica. The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes.

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

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.

Anthranilate phosphoribosyl transferase (TrpD) generates phosphoribosylamine for thiamine synthesis from enamines and phosphoribosyl pyrophosphate.

Anthranilate phosphoribosyl transferase (TrpD) has been well characterized for its role in the tryptophan biosynthetic pathway. Here, we characterized a new reaction catalyzed by TrpD that resulted in the formation of the purine/thiamine intermediate metabolite phosphoribosylamine (PRA). The data showed that 4- and 5-carbon enamines served as substrates for TrpD, and the reaction product was predicted to be a phosphoribosyl-enamine adduct. Isotopic labeling data indicated that the TrpD reaction product was hydrolyzed to PRA. Variants of TrpD that were proficient for tryptophan synthesis were unable to support PRA formation in vivo in Salmonella enterica. These protein variants had substitutions at residues that contributed to binding substrates anthranilate or phosphoribosyl pyrophosphate (PRPP). Taken together the data herein identified a new reaction catalyzed by a well-characterized biosynthetic enzyme, and both illustrated the robustness of the metabolic network and identified a role for an enamine that accumulates in the absence of reactive intermediate deaminase RidA.

Perturbations in histidine biosynthesis uncover robustness in the metabolic network of Salmonella enterica.

Phosphoribosylamine (PRA) is an intermediate in the biosynthetic pathway that is common to thiamine and purines. Glutamine phosphoribosyl pyrophosphate (PRPP) amidotransferase is the product of the purF gene in Salmonella enterica and catalyzes the synthesis of PRA from PRPP and glutamine. Strains lacking PurF require exogenous addition of purines for growth. However, under some growth conditions or with specific secondary mutations these strains grow in the absence of exogenous thiamine. Mutant alleles of hisA, which encodes 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide (ProFAR) isomerase, allowed PurF-independent PRA formation. The alleles of hisA that suppressed the requirement for exogenous thiamine resulted in proteins with reduced enzymatic activity. Data presented here showed that decreased activity of HisA altered metabolite pools and allowed PRA formation from ProFAR. Possible mechanisms of this conversion were proposed. The results herein emphasize the plasticity of the metabolic network and specifically highlight the potential for chemical syntheses to contribute to network robustness.

Suppressor analyses identify threonine as a modulator of ridA mutant phenotypes in Salmonella enterica.

The RidA (YjgF/YER057c/UK114) family of proteins is broadly conserved in the three domains of life yet the functional understanding of these proteins is at an early stage. Physiological studies of ridA mutant strains of Salmonella enterica provided a framework to inform in vitro studies and led to the description of a conserved biochemical activity for this family. ridA mutant strains of S. enterica have characteristic phenotypes including new synthesis of thiamine biosynthetic intermediate phosphoribosylamine (PRA), inability to grow on pyruvate as a sole carbon and energy source or when serine is present in the minimal growth medium, and a decreased specific activity of transaminase B (IlvE). Secondary mutations restoring growth to a ridA mutant in the presence of serine were in dapA (encoding dihydrodipicolinate synthase) and thrA (encoding homoserine dehydrogenase). These mutations suppressed multiple ridA mutant phenotypes by increasing the synthesis of threonine. The ability of threonine to suppress the metabolic defects of a ridA mutant is discussed in the context of recent biochemical data and in vivo results presented here.

Conserved YjgF protein family deaminates reactive enamine/imine intermediates of pyridoxal 5'-phosphate (PLP)-dependent enzyme reactions.

The YjgF/YER057c/UK114 family of proteins is conserved in all domains of life, suggesting that the role of these proteins arose early and was maintained throughout evolution. Metabolic consequences of lacking this protein in Salmonella enterica and other organisms have been described, but the biochemical function of YjgF remained unknown. This work provides the first description of a conserved biochemical activity for the YjgF protein family. Our data support the conclusion that YjgF proteins have enamine/imine deaminase activity and accelerate the release of ammonia from reactive enamine/imine intermediates of the pyridoxal 5'-phosphate-dependent threonine dehydratase (IlvA). Results from structure-guided mutagenesis experiments suggest that YjgF lacks a catalytic residue and that it facilitates ammonia release by positioning a critical water molecule in the active site. YjgF is renamed RidA (reactive intermediate/imine deaminase A) to reflect the conserved activity of the protein family described here. This study, combined with previous physiological studies on yjgF mutants, suggests that intermediates of pyridoxal 5'-phosphate-mediated reactions may have metabolic consequences in vivo that were previously unappreciated. The conservation of the RidA/YjgF family suggests that reactive enamine/imine metabolites are of concern to all organisms.

Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.