title: Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii.
abstract: d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-8308, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hirato
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-8308, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Tokuhisa
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-8308, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Tanigawa
authors.affiliation: Department of Molecular and Functional Genomics, Interdisciplinary Center for Science Research, Shimane University, Nishikawatsu 1060, Matsue, Shimane, 690-8504, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ashida
authors.affiliation: Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Shiga, 520-2192, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-8308, Japan; Department of Biotechnology and Materials Chemistry, Junior College, Nihon University, 7-24-1Narashinodai, Funabashi, Chiba, 274-8501, Japan. Electronic address: nishimura.katsushi@nihon-u.ac.jp.
authors.forename: Katsushi
authors.initials: K
authors.lastname: Nishimura
mesh_terms: D000446:Aldehyde-Lyases / Q000378:metabolism; D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013912:Threonine
chemical_list: D013912:Threonine; D011732:Pyridoxal Phosphate; D012696:Glycine Hydroxymethyltransferase; D000446:Aldehyde-Lyases; C108811:threonine acetaldehyde-lyase; D005998:Glycine
keywords: Chlamydomonadaceae; Chlamydomonas reinhardtii; Enzyme characterization; Green alga; Pyridoxal 5’-phosphate; d-Amino acid; d-Threonine aldolase