title: Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.
abstract: 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(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·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.
authors.affiliation: Department of Applied Chemistry, Waseda University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Arai
authors.forename: Atsushi
authors.initials: A
authors.lastname: Noguchi
authors.forename: Eriko
authors.initials: E
authors.lastname: Takano
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D024342:Amino Acids, Acidic / Q000737:chemistry*; D004151:Dipeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D024342:Amino Acids, Acidic; D004151:Dipeptides; D029701:Saccharomyces cerevisiae Proteins; C432806:glutaminyl-glycine; D000581:Amidohydrolases; C585586:NTA1 protein, S cerevisiae; C019665:amidase; D010453:Peptide Synthases