Loss of YggS (COG0325) impacts aspartate metabolism in Salmonella enterica

Abstract

YggS is a pyridoxal 5'-phosphate (PLP)-binding protein of the conserved COG0325 family. Despite a connection with vitamin B₆ homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP-dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP-dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5'-phosphate and α-keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B₆ vitamers via transamination.

Keywords: YggS; aspartate; pyridoxal 5′-phosphate; transamination; vitamin B6; α-keto acids.

Figure 1.

Aspartate synthesis in S. enterica relies on PLP-dependent transamination reaction. In the first half-reaction of transamination, glutamate donates an amino group to PLP, generating α-ketoglutarate and PMP. In the second half-reaction, PMP is recycled back to PLP, transferring the amino group to oxaloacetate and producing aspartate. PLP is bound to the active site via a Schiff base linkage, while PMP is not covalently bound to the enzyme (indicated by a solid and dashed line, respectively). Oxaloacetate is synthesized from intermediates in glycolysis (PEP, pyruvate) or the TCA cycle (malate, citrate), as depicted. Abbreviations: AspAT, aspartate aminotransferase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; Glu, L-glutamate; αKG, α-ketoglutarate; Asp, L-aspartate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid.

Figure 2.

Loss of yggS leads to aspartate limitation in an aspC background. S. enterica wild-type (DM15847, squares), yggS (DM15948, triangles), aspC (DM16150; circles), and yggS aspC (DM16154; diamonds) strains were grown in minimal NCE with (a) gluconate or (b) pyruvate as a sole carbon source in the absence (white symbols) or presence (gray symbols) of 1.8 mM aspartate. Representative data from three independent experiments, each with three biological replicates, are shown. Error bars depict standard deviation from the mean.

Figure 3.

Suppressors of aspartate requirement in a yggS aspC strain increase TyrB level. (a) DNA sequence of the regulatory region of tyrB, including the −35 and −10 element of the promoter (green), the binding site of the transcriptional regulator TyrR (black box), the ribosomal binding site (pink box), and the start codon of tyrB (orange), is shown. Suppressor mutations linked to tyrB were isolated on minimal NCE glucose (red) or gluconate (blue). The allele in purple was isolated independently on glucose and on gluconate. The number of independent isolates of each substitution is indicated in parentheses. (b) Growth of representative yggS aspC revertants in minimal NCE glucose is depicted. (c) yggS aspC strains carrying empty vector control (pCV1, DM16265), vector expressing yggS (pyggS, DM16470), aspC (paspC, DM16266), or tyrB (ptyrB, DM16267) were grown in minimal NCE gluconate without (white symbols) or with 0.02% arabinose (gray symbols) to induce expression of the respective gene. Data were obtained from three biological replicates from two independent experiments, and error bars show standard deviation from the mean.

Figure 4.

PNP inhibits AspAT activity of TyrB in vitro. (a) After Ni-affinity purification, 2 μg of TyrB-5xHis and AspC-5xHis were separated on 14% SDS-PAGE gel and visualized with Coomassie blue staining. (b) AspAT activity was measured by a coupled assay as the rate of NADH oxidation when oxaloacetate (produced from aspartate and αKG by AspAT) was converted to malate. Assays were performed with (solid bars) or without (open bars) a 10-minute preincubation with PNP prior to initiating the reaction with αKG. Reaction mixtures contained 50 mM HEPES (pH 7.8), 25 nM TyrB or AspC, 100 mM Asp, 0.4 mM NADH, 5U malate dehydrogenase, 5 mM αKG, 2.5 μM PLP, and 0-50 μM PNP. Data were averaged from two independent experiments with at least three technical replicates each. Statistical significance was determined by One-way ANOVA followed by Bonferroni post hoc test for each AspAT. An asterisk indicates adjusted P < 0.0001. Abbreviations: AspAT, aspartate aminotransferase; PNP, pyridoxine 5′-phosphate.

Figure 5.

Exogenous α-keto acids phenocopy a yggS mutation. Growth of wild type (DM15847) and aspC mutant (DM16154) in minimal NCE gluconate in the presence of (a) 1mM αKB or (b) 10 mM dmKG is shown. Addition of 1.8 mM aspartate restored growth of both strains. Average optical density at 650 nm (OD₆₅₀) and standard deviation of three biological replicates from two independent experiments are shown. Abbreviations: αKB, α-ketobutyrate; dmKG, dimethyl α-ketoglutarate; Asp, L-aspartate.