title: Alteration of the substrate specificity of l-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer.
abstract: Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an l-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.
authors.affiliation: a Faculty of Science and Engineering, Department of Applied Chemistry , Waseda University , Tokyo , Japan.|b Technical Research Institute R&D Center , T.Hasegawa Co., Ltd , Kanagawa , Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Kino
authors.affiliation: a Faculty of Science and Engineering, Department of Applied Chemistry , Waseda University , Tokyo , Japan.
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology; D004151:Dipeptides / Q000737:chemistry*; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013649:Taste
chemical_list: D000596:Amino Acids; D004151:Dipeptides; C098230:methionylglycine; D008025:Ligases
keywords: dipeptides; l-amino acid ligase; salt taste enhancer; site-directed mutagenesis