title: L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity.
abstract: L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Qin
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Fabiana Lica
authors.initials: FL
authors.lastname: Imai
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531, Japan.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kataoka
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Nahoko
authors.initials: N
authors.lastname: Kitamura
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Urano
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Mori
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawabata
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, 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-ku, Tokyo 113-8657, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtsuka
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Feng
authors.initials: F
authors.lastname: Hou
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D000333:Aeromonas / Q000201:enzymology*; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D012696:Glycine Hydroxymethyltransferase
keywords: l-allo-threonine aldolase; pyridoxal 5′-phosphate; stereoselectivity; α/β domain