title: Engineering an ATP-dependent D-Ala:D-Ala ligase for synthesizing amino acid amides from amino acids.
abstract: We successfully engineered a new enzyme that catalyzes the formation of D-Ala amide (D-AlaNH2) from D-Ala by modifying ATP-dependent D-Ala:D-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of D-Ala-D-Ala from two molecules of D-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second D-Ala of D-Ala-D-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for D-AlaNH2production. The S293E variant, which was selected as the best enzyme for D-AlaNH2production, exhibited an optimal activity at pH 9.0 and 40 °C for D-AlaNH2production. The apparent Kmvalues of this variant for D-Ala and NH3were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of D-AlaNH2from 10 and 50 mM D-Ala and 3 M NH4Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.
authors: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.|Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.|MicroBiopharm Japan Co.Ltd., 1-3-1 Kyobashi, Chuo-ku, Tokyo, 104-0031, Japan.
authors: Yuta
authors: Y
authors: Miki
authors: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.|Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan.
authors: Seiji
authors: S
authors: Okazaki
authors: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. asano@pu-toyama.ac.jp.|Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan. asano@pu-toyama.ac.jp.
authors: Yasuhisa
authors: 0000-0003-3645-3952
authors: Y
authors: Asano
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000577:Amides / Q000737:chemistry* / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics
chemical_list: D000577:Amides; D000596:Amino Acids; D004151:Dipeptides; C002956:alanylalanine; D000255:Adenosine Triphosphate; D008025:Ligases
keywords: Amino acid; Amino acid amide; D-Ala:D-Ala ligase; Docking simulation; Homology modeling; Protein engineering