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

Abstract

Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64% identity with LkAlt1 and 66% with KlAlt1, suggests that ScAlt2 diversified after the ancestral hybrid was formed. ScALT2 functional diversification resulted in loss of both alanine transaminase activity and the additional alanine-independent LkAlt1 function, since ScALT2 did not complement the Lkalt1Δ phenotype. It can be concluded that LkALT1 and KlLALT1 functional role as alanine transaminases was delegated to ScALT1, while ScALT2 lost this role during diversification.

Saccharomyces cerevisiae Differential Functionalization of Presumed ScALT1 and ScALT2 Alanine Transaminases Has Been Driven by Diversification of Pyridoxal Phosphate Interactions

“…In the case of the Sc Alt1, its previously described role as alanine transaminase ( Peñalosa-Ruiz et al, 2012 ) was confirmed, and its kinetic characterization showed that it has the characteristic ping-pong mechanism displayed by transaminases. It was additionally found that the kinetic parameters of Sc Alt1 are similar to those reported for the orthologous proteins Kl Alt1 and Lk Alt1 ( Escalera-Fanjul et al, 2017 ).…”

“…The resulting kinetic parameters are presented in Table 1 . In addition, Table 1 shows the kinetic parameters for two ancestral type yeasts: Lk Alt1 ( Lacchancea kluyveri ) and Kl Alt1 ( Kluyveromyces lactis ) ( Escalera-Fanjul et al, 2017 ). Sc Alt1 showed a Km for alanine and α-ketoglutarate similar to those found for Lk Alt1 and Kl Alt1, while Sc Alt1 k cat was two to threefold higher than the ones found in both of the ancestral-type species, this could be attributed to the fact that for Sc Alt1, purification was carried at 4°C, while those of Lk Alt1 and Kl Alt1 were performed at room temperature (25°C).…”

“…Although these paralogs have diversified in various ways, both of them have been retained in the yeast S. cerevisiae genome for over 100 million years, although it has been observed that the half-life of a duplicated eukaryotic gene is of approximately 4 million years ( Lynch and Conery, 2000 ). ScALT1 encodes an alanine transaminase, which conserved the ancestral capacity to biosynthesize alanine and has specialized its catabolic role, constituting an indispensable enzyme for alanine utilization as sole nitrogen source ( Escalera-Fanjul et al, 2017 ). Conversely, ScAlt2 protein has completely lost its role in alanine metabolism ( Peñalosa-Ruiz et al, 2012 ).…”

“…Accordingly, it has been proposed that in S. cerevisiae , the action of the glutamine-pyruvate transaminase and the ω-amidase pathway could constitute an alternative alanine biosynthetic route ( Soberón et al, 1989 ), that would not play a catabolic role, since the reaction catalyzed by glutamine transaminase is irreversible ( Soberón et al, 1989 ). In regard to ancestral-type yeasts, previous results from our laboratory ( Escalera-Fanjul et al, 2017 ) have shown that Lacchancea kluyveri ( L. kluyveri ) and Kluyveromyces lactis ( K. lactis ) alanine transaminases; LkAlt1 and KlAlt1 are ScAlt1 orthologous counterpart. Additionally, these two yeasts respectively display Lk Alt1 and Kl Alt1 alanine transaminase activity and unidentified alanine biosynthetic and catabolic pathways.…”

“…In addition, the phenotypic analysis of Lkalt1 Δ and Klalt1 Δ null mutants showed that these proteins have a metabolic role, not related to alanine metabolism, since Lkalt1 Δ does not achieve wild type growth rate. Thus, the ancestral alanine transaminase function ( Escalera-Fanjul et al, 2017 ) was kept by the ScAlt1 , which specialized its catabolic character, and lost the alanine independent role observed in the ancestral type enzymes. The fact that Sc Alt2 and Sc Alt1 conserve high identity with Lk Alt1 and Kl Alt1, indicates that Sc Alt1 and Sc Alt2 diversified after the ancestral hybrid was formed.…”