Isolation of a Mutant Auxotrophic for<scp>L</scp>-Alanine and Identification of Three Major Aminotransferases That Synthesize<scp>L</scp>-Alanine in<i>Escherichia coli</i>

Yoneyama, Hiroshi; Hori, Hatsuhiro; Lim, Sang Jo; Murata, Tatsuya; Ando, Tasuke; Isogai, Emiko; Katsumata, Ryoichi

doi:10.1271/bbb.100905

Cited by 15 publications

(14 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, increased catabolic character did not affect the prominent biosynthetic role played by these three enzymes, which contribute with at least half of the alanine pool, even when more than one pathway for alanine biosynthesis is present in the three species. This redundancy is also found in E. coli , where alanine is synthesized by three different transaminases, and only the triple mutant is an alanine auxotroph (Yoneyama et al, 2011). In this regard, it is important to consider that in E. coli alanine is the second amino acid with highest intracellular concentration, and in Neurospora and S. cerevisiae it is the amino acid displaying highest intracellular concentration (Yuan et al, 2006; Canelas et al, 2008; Kim et al, 2011).…”

Section: Discussionmentioning

confidence: 87%

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

et al. 2017

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 87%

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

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Auxotrophic E. coli mutants for L‐alanine ( HYE032 ) ( avtA::GM, yfbQ::KM, yfdZ::FRT, Ala − ) were obtained from Dr. Hiroshi Yoneyama from Tohoku University (Yoneyama et al, ). The E. coli mutant auxotrophic for β‐alanine ( panD ) (F‐, Δ(araD‐araB)567 , ΔpanD748::kan , ΔlacZ4787c (::rrnB‐3), λ − , rph‐1 , Δ(rhaD‐rhaB)568 , hsdR514 ) was obtained from the Coli Genetic Stock Center (CGSC #8404) (http://cgsc2.biology.yale.edu/) (Baba et al, ).…”

Section: Methodsmentioning

confidence: 99%

The Arabidopsis thaliana gene annotated by the locus tag At3g08860 encodes alanine aminotransferase

et al. 2019

View full text Add to dashboard Cite

The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes, eight of which are suggested to be alanine aminotransferases. One of the putative alanine aminotransferases genes, At3g08860, was attributed the function of alanine:glyoxylate aminotransferase/β‐alanine:pyruvate aminotransferase based on the analysis of gene expression networks and homology to other β‐alanine aminotransferases in plants. It was earlier demonstrated that At3g08860 is specifically upregulated in response to osmotic stress, but not other stresses (β‐alanine is an osmoprotectant in plants). Furthermore, it was shown that the expression of At3g08860 is highly coordinated with the genes of the uracil degradation pathway leading to the non‐proteinogenic amino acid β‐alanine. These evidence were suggestive of the involvement of At3g08860 in β‐alanine metabolism. However, direct experimental evidence for the function of At3g08860 was lacking, and therefore, the goal of this study was to elucidate the function of the uncharacterized aminotransferase annotated by the locus tag At3g08860. The cDNA of At3g08860 was demonstrated to functionally complement two E. coli mutants auxotrophic for the amino acids, L‐alanine (proteinogenic) and β‐alanine (non‐proteinogenic). Enzyme activity using purified recombinant At3g08860 further demonstrated that the enzyme is endowed with L‐alanine:glyoxylate aminotransferase activity.

show abstract

“…For instance, the aspartate aminotransferase, tyrosine aminotransferase, and the branched-chain amino acid aminotransferase have been shown to possess overlapping activities in E. coli (Gu et al, 1998). In addition, a recent study demonstrated that three aminotransferases are involved in alanine biosynthesis in E. coli (Yoneyama et al, 2011). …”

Section: Discussionmentioning

confidence: 99%

Genomic and Biochemical Analysis of the Diaminopimelate and Lysine Biosynthesis Pathway in Verrucomicrobium spinosum: Identification and Partial Characterization of L,L-Diaminopimelate Aminotransferase and UDP-N-Acetylmuramoylalanyl-D-glutamyl-2,6-meso-Diaminopimelate Ligase

et al. 2012

View full text Add to dashboard Cite

The Gram-negative bacterium Verrucomicrobium spinosum has attracted interest in recent years following the sequencing and annotation of its genome. Comparative genomic analysis of V. spinosum using diaminopimelate/lysine metabolic genes from Chlamydia trachomatis suggests that V. spinosum employs the L,L-diaminopimelate aminotransferase (DapL) pathway for diaminopimelate/lysine biosynthesis. The open reading frame corresponding to the putative dapL ortholog was cloned and the recombinant enzyme was shown to possess L,L-diaminopimelate aminotransferase activity in vitro. In vivo analysis using functional complementation confirmed that the dapL ortholog was able to functionally complement an E. coli mutant that confers auxotrophy for diaminopimelate and lysine. In addition to its role in lysine biosynthesis, the intermediate diaminopimelate has an integral role in peptidoglycan biosynthesis. To this end, the UDP-N-acetylmuramoylalanyl-d-glutamyl-2,6-meso-diaminopimelate ligase ortholog was also identified, cloned, and was shown to possess meso-diaminopimelate ligase activity in vivo. The L,L-diaminopimelate aminotransferase pathway has been experimentally confirmed in several bacteria, some of which are deemed pathogenic to animals. Since animals, and particularly humans, lack the genetic machinery for the synthesis of diaminopimelate/lysine de novo, the enzymes involved in this pathway are attractive targets for development of antibiotics. Whether dapL is an essential gene in any bacteria is currently not known. V. spinosum is an excellent candidate to investigate the essentiality of dapL, since the bacterium employs the DapL pathway for lysine and cell wall biosynthesis, is non-pathogenic to humans, facile to grow, and can be genetically manipulated.

show abstract

Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

Cited by 15 publications

References 41 publications

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

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

The Arabidopsis thaliana gene annotated by the locus tag At3g08860 encodes alanine aminotransferase

Genomic and Biochemical Analysis of the Diaminopimelate and Lysine Biosynthesis Pathway in Verrucomicrobium spinosum: Identification and Partial Characterization of L,L-Diaminopimelate Aminotransferase and UDP-N-Acetylmuramoylalanyl-D-glutamyl-2,6-meso-Diaminopimelate Ligase

Contact Info

Product

Resources

About