Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23.
The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.
Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan.
Emran Kabir
EK
Chowdhury
Yuka
Y
Akaishi
Shinji
S
Nagata
Haruo
H
Misono
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C021398:3-hydroxyisobutyrate dehydrogenase