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

Abstract

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.

FIGURE 1.

Threonine dehydration and YjgF function. IlvA dehydrates threonine to generate enamine/imine tautomer intermediates. The imine is hydrolyzed non-enzymatically to form the final products 2-ketobutyrate and ammonia. The data reported under “Results” showed that a biochemical function of YjgF is to catalyze the hydrolysis of these intermediates, thus removing enamine/imine from solution and increasing the rate of production of 2-ketobutyrate.

FIGURE 2.

Presence of YjgF changes saturation curve of IlvA. The initial rate of 2-ketobutyrate (2KB) formation versus threonine concentration ([Thr]); IlvA alone (squares); and IlvA + YjgF (circles) were measured. A, pH 7.5; B, pH 9.5. Error bars represent S.E. of two replicates; these data are representative of more than five independent experiments.

FIGURE 3.

IlvA and YjgF have different pH profiles. The initial rate of 2-ketobutyrate (2KB) formation from 15 mm threonine versus pH; IlvA alone (squares) and IlvA + YjgF (circles) was determined. Buffers are 50 mm each MES/HEPES/TAPS (pH ≤ 8.6; open symbols) or 50 mm each TAPS/CHES/CAPS (pH ≥ 8.3; closed symbols). Error bars represent S.D. of two replicates. These data are representative of two independent experiments.

FIGURE 4.

YjgF decreases availability of enamine/imine intermediates of IlvA. A, ferricyanide reduction was followed by absorbance at 420 nm over time. Assays contained 10 mm threonine, 5 mm ferricyanide, and either IlvA alone (dotted line) or IlvA with YjgF (solid line). B, quantities of ferricyanide reduced (gray bars) and 2-ketobutyrate produced (white bars) in the presence and absence of YjgF after 2 h, with 10 mm threonine and 0, 1, 2, or 5 mm ferricyanide (FeCN) as indicated. Ferricyanide concentrations were determined by A_{420 nm}, and 2-ketobutyrate concentrations were determined by 2,4-dinitrophenylhydrazine derivatization (see ”Experimental Procedures“). Error bars represent S.D. of two replicates; these data are representative of nine and four independent experiments (A and B, respectively).

FIGURE 5.

Alignment of family members and proposed mechanism for YjgF. A, amino acid sequences of YjgF family members assayed in this study were aligned using CLUSTALW (45). The numbering of residues is based on the S. enterica YjgF sequence. YjgF is from S. enterica; YabJ is from B. subtilis; PF0668 is from P. furiosus; ChrD is from C. sativus; UK114 is from H. sapiens; and RutC is from E. coli K12. Boxed residues in the S. enterica sequence indicate amino acids mutated in this study. Asterisks indicate residues that are invariant among high identity family members (9) and have been predicted to be involved in active site structure or catalysis (5). B, a reaction scheme for the deamination of iminobutyrate by YjgF. The backbone of Cys-107 and the side chain of Glu-120 hydrogen-bond with water, whereas the side chain of Arg-105 forms a salt bridge with the carboxyl group of the substrate and the Tyr-17 hydroxyl group hydrogen bonds with the imine nitrogen. This setup facilitates the attack of water on C2 of the imine. Then a rearrangement occurs, releasing the products ammonia and 2-ketobutyrate.

FIGURE 6.

Activity of YjgF variants. Standard assays contained 15 mm threonine in 50 mm HEPES, pH 8.0 (white bars) or CHES, pH 9.5 (gray bars), with 0.9 μm IlvA and the indicated YjgF protein (all variants present at 1.6 μm). Activity of YjgF is represented as the -fold increase in the rate of 2-ketobutyrate formation over the rate of IlvA alone (set to 1). Error bars represent S.D. of three replicates. Data are representative of three independent experiments.

FIGURE 7.

A serine-derived metabolite is another substrate for YjgF. Initial rate of pyruvate (Pyr) formation versus serine concentration ([Ser]) at pH 9.5; IlvA alone (squares) and IlvA + YjgF (circles) were measured. Error bars represent S.E. of two replicates. Data are representative of three independent experiments.

FIGURE 8.

Deaminase activity of YjgF is conserved across domains. Initial rate of 2-ketobutyrate (2KB) formation versus threonine concentration ([Thr]) at pH 8.0 with diverse YjgF homologs (all present at 1.6 μm). IlvA alone (closed squares), IlvA + S. enterica YjgF (closed circles), IlvA + B. subtilis YabJ (closed triangles), IlvA + H. sapiens UK114 (open squares), IlvA + C. sativus ChrD (open triangles), IlvA + P. furiosus PF0668 (open diamonds), and IlvA + E. coli RutC (open circles) were measured. Error bars represent S.E. of two replicates. These data are representative of two independent experiments.