Cloning, sequencing, and high level expression of the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae

Takamasa Tobimatsu, Muneaki Azuma, Hirokazu Matsubara, Hiroe Takatori, Takashi Niida, Kyouichi Nishimoto, Hideaki Satoh, Ryuji Hayashi, Tetsuo Toraya

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70 Citations (Scopus)

Abstract

The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes. Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed. The enzyme expressed in E. coli was indistinguishable from the wild-type glycerol dehydrase of K. pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties. It was also shown that the recombinant functional enzyme consists of M(r) 61,000, 22,000, and 16,000 subunits. Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases. The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(α), 21,355(β), and 16,104(γ), respectively. High level expression of these three genes in E. coli produced more than 14-fold higher level of fully active apoenzyme than that in K. pneumoniae. It was thus concluded that these are the genes encoding the subunits of glycerol debydrase. The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the α, β, and γ subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins.

Original languageEnglish
Pages (from-to)22352-22357
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number37
DOIs
Publication statusPublished - Sep 25 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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