title: Purification and characterization of alanine dehydrogenase from a cyanobacterium, Phormidium lapideum.
abstract: Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0 mM for L-alanine and 0.04 mM for NAD+, 0.33 mM for pyruvate, 60.6 mM for NH4+ (pH 8.7), and 0.02 mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP.
authors.affiliation: Department of Applied Biochemistry, Faculty of Agriculture, Shimane University.
authors.forename: Y
authors.initials: Y
authors.lastname: Sawa
authors.forename: M
authors.initials: M
authors.lastname: Tani
authors.forename: K
authors.initials: K
authors.lastname: Murata
authors.forename: H
authors.initials: H
authors.lastname: Shibata
authors.forename: H
authors.initials: H
authors.lastname: Ochiai
mesh_terms: D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003067:Coenzymes / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase