Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC-1: purification, molecular characterization and expression.
We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be approximately 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of alpha-ketoisovalerate, alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketocaproate. The reaction proceeds through an iso-ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 degrees C was estimated to be 2.4 min. The kcat/Km (micro(-1).s(-1)) values for L-valine and NAD+ at 20 degrees C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.
Oikawa
T
T
Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka-Fu, Japan.
Yamanaka
K
K
Kazuoka
T
T
Kanzawa
N
N
Soda
K
K
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D003589:Cytophaga / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D000594:Amino Acid Oxidoreductases; C057545:valine dehydrogenase (NAD+)