TY - JOUR
T1 - Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca
AU - Tobimatsu, Takamasa
AU - Sakai, Tadahiro
AU - Hashida, Yasuharu
AU - Mizoguchi, Nika
AU - Miyoshi, Shozo
AU - Toraya, Tetsuo
N1 - Funding Information:
1This work was supported in part by Grants-in-Aid for Scienti®c Research on Priority Areas (Molecular Biometallics, No. 07229234 and No. 08249226) from the Ministry of Education, Science, Sports, and Culture, Japan, and research grants from Nagase Science and Technology Foundation, from Okayama Foundation for Science and Technology, and from the Japan Society for the Promotion of Science (Research for the Future, RFTF96L00506).
PY - 1997/11/1
Y1 - 1997/11/1
N2 - Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65% The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K. oxytoca. The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparent M(r) of 60,000 (α), 30,000 (β), and 19,000 (γ). Therefore, the subunit structure of the enzyme is most likely α2β2γ2. The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate. Components F and S were identified as the β subunit and α2γ2 complex, respectively. Apparent K(m) for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2 ethanediol were 0.83 μM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively The three genes encoding the sub- units of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The α and γ subunits mutually required each other for correct folding forming the soluble, active α2γ2 complex (component S). Expression of the β subunit in a soluble, active form (component F) was promoted by coexpression with both the α and γ subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme.
AB - Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65% The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K. oxytoca. The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparent M(r) of 60,000 (α), 30,000 (β), and 19,000 (γ). Therefore, the subunit structure of the enzyme is most likely α2β2γ2. The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate. Components F and S were identified as the β subunit and α2γ2 complex, respectively. Apparent K(m) for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2 ethanediol were 0.83 μM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively The three genes encoding the sub- units of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The α and γ subunits mutually required each other for correct folding forming the soluble, active α2γ2 complex (component S). Expression of the β subunit in a soluble, active form (component F) was promoted by coexpression with both the α and γ subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme.
KW - Adenosylcobalamin
KW - Diol dehydratase
KW - Recombinant, purification, gene expression
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U2 - 10.1006/abbi.1997.0325
DO - 10.1006/abbi.1997.0325
M3 - Article
C2 - 9344474
AN - SCOPUS:0031282402
VL - 347
SP - 132
EP - 140
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 1
ER -