Paralogous ALT1 and ALT2 Retention and Diversification Have Generated Catalytically Active and Inactive Aminotransferases in Saccharomyces cerevisiae

Abstract

BackgroundGene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase.Principal FindingsOur results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function.Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.

Protein Moonlighting Revealed by Noncatalytic Phenotypes of Yeast Enzymes

“…Likewise, more phenotypes were scored in the alt1Dalt2D double knockout compared to the single alt1D. Intriguingly, the exposed phenotypes were mostly noncatalytic; this is consistent with a lack of catalytic activity in the Alt2 paralog (Peñalosa-Ruiz et al 2012). These results suggest that a moonlighting noncatalytic function is present in Alt1 and shared with its paralog.…”

Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

“…Interestingly, the S. cerevisiae isozyme encoded by ScALT1 , retained alanine transaminase activity, while ScALT2 encoded enzyme did not conserve this activity (Peñalosa-Ruiz et al, 2012). In all three cases transaminase activity followed gene expression levels of their encoding genes.…”

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine

“…It was also proposed that L-alanine can be converted to β-alanine via the action of a non-natural 2,3-alanine aminomutase (AAM) developed by directed evolution of L-lysine 2,3-aminomutase from Bacillus subtilis (Liao et al, 2010). The S. cerevisiae genome contains two genes encoding alanine aminotransferase enzymes, ALT1 and ALT2, of which the latter is catalytically inactive (Peñalosa-Ruiz et al, 2012). We tested the effect of ALT1 or BsAAM overexpression on 3HP production.…”