title: Construction and characterization of hybrid dehydratases between adenosylcobalamin-dependent diol and glycerol dehydratases.
abstract: 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.
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sakai
authors.forename: Ai
authors.initials: A
authors.lastname: Yamasaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Toyofuku
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Nishiki
authors.forename: Michio
authors.initials: M
authors.lastname: Yunoki
authors.forename: Noriaki
authors.initials: N
authors.lastname: Komoto
authors.forename: Takamasa
authors.initials: T
authors.lastname: Tobimatsu
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Toraya
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D003038:Cobamides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C020671:glycerol dehydratase; C000913:cobamamide; D005990:Glycerol