Sequence analysis of the gene for and characterization of d-acetoin forming meso-2,3-butanediol dehydrogenase of Klebsiella pneumoniae expressed in Escherichia coli

Abstract

Analysis of the nucleotide sequence of the meso-2,3-butanediol dehydrogenase ( d-acetoin forming) (BDH) gene of Klebsiella pneumoniae IAM1063 revealed that it contains an open reading frame of 768 bp encoding a polypeptide of 256 amino acid residues with a molecular weight of 26,591 Da. The amino acid sequence deduced from the nucleotide sequence of the BDH gene is consistent with the amino-terminal and carboxyl-terminal amino acid sequences, amino acid composition, and molecular weight of purified BDH. The amino acid sequences of BDHs from K. pneumoniae IAM1063 and Klebsiella terrigena VTT-E-74023 showed 92.9% homology in the first 196 amino acid residues, but no homology elsewhere. Moreover, the BDH of K. pneumoniae IAM1063 was 15 amino acid residues longer than that of K. terrigena VTT-E-74023. The characteristics of the BDH from Escherichia coli JM109 pBUD119 (containing the meso-BDH gene) coincided with that of the BDH from the gene source strain ( K. pneumoniae IAM1063). Furthermore, the BDHs from K. pneumoniae IAM1063 and K. terrigena VTT-E-74023 showed similar catalytic properties. This suggests that the downstream of Gln at position 196 in the amino acid sequence is unrelated to BDH activity.