title: Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1.
abstract: We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123 k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K(m) (microM(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Kansai University, Osaka, Japan. oikawa@ipcku.kansai-u.ac.jp
authors.forename: Tadao
authors.initials: T
authors.lastname: Oikawa
authors.forename: Noriko
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Koji
authors.initials: K
authors.lastname: Shimoke
authors.forename: Shinichi
authors.initials: S
authors.lastname: Uesato
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ikeuchi
authors.forename: Toru
authors.initials: T
authors.lastname: Fujioka
mesh_terms: D000864:Antarctic Regions; D002384:Catalysis; D003080:Cold Temperature; D004795:Enzyme Stability; D005417:Flavobacterium / Q000201:enzymology*; D006358:Hot Temperature; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009249:NADP; D010071:Oxaloacetates / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D008293:Malates; D010071:Oxaloacetates; D009249:NADP; C030298:malic acid; D008291:Malate Dehydrogenase