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The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF , transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools.

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.

Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (2 days) at ambient temperature or short incubations (10 min) ator =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.