title: Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.
abstract: Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Toraya
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Tanokuchi
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Yamasaki
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ogura
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Tobimatsu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003038:Cobamides / Q000378:metabolism*; D004789:Enzyme Activation; D005486:Flavin Mononucleotide / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D041121:Klebsiella oxytoca / Q000201:enzymology / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D011408:Propanediol Dehydratase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003038:Cobamides; D006873:Hydroquinones; C540087:flavin mononucleotide hydroquinone; D009243:NAD; D005486:Flavin Mononucleotide; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; C019920:cob(I)alamin adenosyltransferase; D011408:Propanediol Dehydratase; C000913:cobamamide; D008274:Magnesium