title: Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1.
abstract: 2,4-Diaminopentanoate dehydrogenase (2,4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R,4S)-2,4-diaminopentanoate (2,4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2,4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2,4-DAP, indicating that the protein is a 2,4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2,4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2,4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2,4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2,4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2,4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2,5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway.
authors: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors: Sadanobu
authors: S
authors: Fukuyama
authors: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors: Hisaaki
authors: H
authors: Mihara
authors: Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama 227-8502, Japan; API Corporation, Yokohama 227-8502, Japan.
authors: Ryoma
authors: R
authors: Miyake
authors: Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama 227-8502, Japan; API Corporation, Yokohama 227-8502, Japan.
authors: Makoto
authors: M
authors: Ueda
authors: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors: Nobuyoshi
authors: N
authors: Esaki
authors: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Electronic address: kurihara@scl.kyoto-u.ac.jp.
authors: Tatsuo
authors: T
authors: Kurihara
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000599:Amino Acids, Diamino / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009952:Ornithine / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: C000590799:2,5-diaminohexanoate; D000599:Amino Acids, Diamino; D011994:Recombinant Proteins; D009952:Ornithine; D000594:Amino Acid Oxidoreductases; C020030:2,4-diaminopentanoic acid dehydrogenase
keywords: 2,4-Diaminopentanoate dehydrogenase; Amination; Amino acid dehydrogenase; Deamination; Fervidobacterium nodosum; Ornithine metabolism; Thermophilic enzyme