Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca.
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 Mr of 60,000 (alpha), 30,000 (beta), and 19,000 (gamma). Therefore, the subunit structure of the enzyme is most likely alpha2beta2gamma2. 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 beta subunit and alpha2gamma2 complex, respectively. Apparent Km for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 microM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively. The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The alpha and gamma subunits mutually required each other for correct folding forming the soluble, active alpha2gamma2 complex (component S). Expression of the beta subunit in a soluble, active form (component F) was promoted by coexpression with both the alpha and gamma 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.
Tobimatsu
T
T
Faculty of Engineering, Okayama University, Okayama, Tsushima-Naka, 700, Japan.
Sakai
T
T
Hashida
Y
Y
Mizoguchi
N
N
Miyoshi
S
S
Toraya
T
T
D002852:Chromatography, Ion Exchange; D003038:Cobamides / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D007709:Klebsiella / Q000737:chemistry / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011408:Propanediol Dehydratase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis
D003038:Cobamides; D003902:Detergents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000077423:Polidocanol; D011092:Polyethylene Glycols; D011408:Propanediol Dehydratase; C000913:cobamamide