title: L-Arginine oxidase from Pseudomonas sp. TPU 7192: Characterization, gene cloning, heterologous expression, and application to L-arginine determination.
abstract: L-Arginine oxidase (AROD, EC 1.4.3.-) was discovered in newly discovered Pseudomonas sp. TPU 7192 and its characteristics were described. The molecular mass (MS) of the enzyme was estimated to be 528 kDa, which was accounted for by eight identical subunits with MS of 66 kDa each. AROD was identified as a flavin adenine dinucleotide (FAD)-dependent enzyme with 1 mol of FAD being contained in each subunit. It catalyzed the oxidative deamination of L-arginine and converted L-arginine to 2-ketoarginine, which was non-enzymatically converted into 4-guanidinobutyric acid when the hydrogen peroxide (H2O2) formed by L-arginine oxidation was not removed. In contrast, 2-ketoarginine was present when H2O2was decomposed. AROD was specific to L-arginine with a Km value of 149 μM. It exhibited maximal activity at 55 °C and pH 5.5. AROD was stable in the pH range 5.5-7.5 and >95% of its original activity was below 60 °C at pH 7.0. Since these enzymatic properties are considered suitable for the determination of L-arginine, the gene was cloned and expressed in a heterologous expression system. We herein successfully developed a new simple enzymatic method for the determination of L-arginine using Pseudomonas AROD.
authors.affiliation: 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.forename: Daisuke
authors.initials: D
authors.lastname: Matsui
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Anna
authors.initials: A
authors.lastname: Terai
authors.affiliation: 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. Electronic address: asano@pu-toyama.ac.jp.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015374:Biosensing Techniques; D002172:Canavanine / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D006146:Guanidines / Q000378:metabolism; D006709:Homoarginine / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: C093466:2-ketoarginine; D001426:Bacterial Proteins; D006146:Guanidines; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D006709:Homoarginine; C001317:gamma-guanidinobutyric acid; D002172:Canavanine; D001120:Arginine; D006861:Hydrogen Peroxide; D000594:Amino Acid Oxidoreductases; C040983:arginine oxidase; D008239:Lysine
keywords: 2-Ketoarginine; 4-Guanidinobutyric acid; Flavin adenine dinucleotide; Pseudomonas; l-Arginine determination; l-Arginine oxidase