Construction and characterization of hybrid dehydratases between adenosylcobalamin-dependent diol and glycerol dehydratases.
Adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase are isofunctional enzymes that catalyze the dehydration of 1,2-diols to the corresponding aldehydes. Although they bear different metabolic roles, both enzymes consist of three different subunits and possess a common (alphabetagamma)2 structure. To elucidate the roles of each subunit, we constructed expression plasmids for the hybrid dehydratases between diol dehydratase of Klebsiella oxytoca and glycerol dehydratase of Klebsiella pneumoniae in all the combinations of subunits by gene engineering techniques. All of the hybrid enzymes were produced in Escherichia coli at high levels, but only two hybrid enzymes consisting of the alpha subunit from glycerol dehydratase and the beta subunits from diol dehydratase showed high activity. The substrate specificity, the susceptibility to inactivation by glycerol, and the monovalent cation specificity of the wild type and hybrid enzymes were primarily determined by the origin of their alpha subunits.
Sakai
Takahiro
T
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan.
Yamasaki
Ai
A
Toyofuku
Satoshi
S
Nishiki
Tsuneo
T
Yunoki
Michio
M
Komoto
Noriaki
N
Tobimatsu
Takamasa
T
Toraya
Tetsuo
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003038:Cobamides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005990:Glycerol / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D041121:Klebsiella oxytoca / Q000737:chemistry / Q000201:enzymology; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000201:enzymology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003038:Cobamides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C020671:glycerol dehydratase; C000913:cobamamide; D005990:Glycerol