Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23 |
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Authors: | Chowdhury Emran Kabir Akaishi Yuka Nagata Shinji Misono Haruo |
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Institution: | Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan. |
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Abstract: | 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. |
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