Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ? 30 nmol min mg?protein and 136 ? 6 nmol min mg?protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|