期刊
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
卷 69, 期 3, 页码 455-462出版社
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.69.455
关键词
diol dehydratase; glycerol dehydratase; solubility of enzyme; adenosylcobalamin; coenzyme B-12
Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterobexamer (alpha beta gamma)(2), and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the beta and gamma subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the beta and gamma subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the beta and gamma subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both beta and gamma subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.
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