title: Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination.
abstract: Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2-7.5 and 45 °C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues (112)ML(113), (141)FADTWG(146) M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 °C) gave K m and k cat values of 0.848 ± 0.009 mM and 9.11 ± 0.13 s(-1), respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 μg mL(-1)-2.5 mg mL(-1) (15 μM-3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.
authors.affiliation: Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima, 960-1296, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hirano
authors.affiliation: Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima, 960-1296, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Chonan
authors.affiliation: Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo, Aoba, Sendai, 980-8575, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Murayama
authors.affiliation: Asahi Kasei Pharma Corp, 632-1 Mifuku, Izunokuni, Shizuoka, 410-2321, Japan.
authors.forename: Shin-Ich
authors.initials: SI
authors.lastname: Sakasegawa
authors.affiliation: Asahi Kasei Pharma Corp, 632-1 Mifuku, Izunokuni, Shizuoka, 410-2321, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Matsumoto
authors.affiliation: Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima, 960-1296, Japan. sugimori@sss.fukushima-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sugimori
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019856:Ethanolamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009090:Mucorales / Q000201:enzymology*; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D019856:Ethanolamine; D006631:Amine Oxidase (Copper-Containing)
keywords: Characterization; Cloning; Cu amine oxidase; Enzymatic determination of ethanolamine; Heterologous expression; Syncephalastrum racemosum