title: A new target region for changing the substrate specificity of amine transaminases.
abstract: (R)-stereospecific amine transaminases (R-ATAs) are important biocatalysts for the production of (R)-amine compounds in a strict stereospecific manner. An improved R-ATA, ATA-117-Rd11, was successfully engineered for the manufacture of sitagliptin, a widely used therapeutic agent for type-2 diabetes. The effects of the individual mutations, however, have not yet been demonstrated due to the lack of experimentally determined structural information. Here we describe three crystal structures of the first isolated R-ATA, its G136F mutant and engineered ATA-117-Rd11, which indicated that the mutation introduced into the 136(th) residue altered the conformation of a loop next to the active site, resulting in a substrate-binding site with drastically modified volume, shape, and surface properties, to accommodate the large pro-sitagliptin ketone. Our findings provide a detailed explanation of the previously reported molecular engineering of ATA-117-Rd11 and propose that the loop near the active site is a new target for the rational design to change the substrate specificity of ATAs.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Guan
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtsuka
authors.affiliation: 1] Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan [2] Department of Ocean Sciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Okai
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Mase
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Zhi
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Feng
authors.initials: F
authors.lastname: Hou
authors.affiliation: Biotechnology Development Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Ito
authors.affiliation: Research &Development Group, QOL Division, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Iwasaki
authors.affiliation: Biotechnology Development Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Yasohara
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D000588:Amines / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000588:Amines; D000637:Transaminases