Genetics and Regulation of the Major Enzymes of Alanine Synthesis in Escherichia coli

Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with K_ms for pyruvate within the intracellular range of pyruvate concentration.The enzymes and pathway of alanine synthesis in the model organism Escherichia coli have not been well characterized (25). The most likely pathway is transamination of pyruvate by glutamate, catalyzed by glutamic-pyruvic transaminase (GPT). However, labeling studies have suggested some unanticipated complexities (7, 25, 26). Claire Berg and colleagues performed the only genetic analysis of alanine synthesis. They identified three genes that participate in alanine synthesis, namely, avtA, alaA, and alaB (1, 2, 36, 40). The activity of AvtA, also called transaminase C, was initially detected as an alanine-synthesizing enzyme with valine, not glutamate, as the nitrogen donor (27). Loss of either avtA or alaA did not affect growth and was apparent only in an ilvE background (2, 36, 40). An alaA mutant had normal AvtA and GPT activities, which suggested that AlaA was not a transaminase (1, 36). The alaA gene was physically mapped, but its product was not subsequently characterized (1). The alaB gene was identified from its partial suppression of the phenotype of an ilvE alaA strain (36). Multicopy alaB had elevated GPT activity, which suggested that alaB specifies a GPT (36). Except for a partial physical map of the alaB region, nothing else is known about alaB and its product (36).Our goal in this study was to determine the enzymes of alanine synthesis using current knowledge of known and potential transaminase genes. Our genetic analysis suggests that AvtA, YfbQ, and YfdZ are the major enzymes of alanine synthesis, but eight other transaminases can potentially synthesize alanine. To confirm these conclusions, we also analyzed the regulation of avtA, yfbQ, and yfdZ and purified and partially characterized YfbQ and YfdZ.