title: Gene cloning and biochemical characterization of a catalase from Gluconobacter oxydans.
abstract: Gluconobacter oxydans has a large number of membrane-bound dehydrogenases linked to the respiratory chain that catalyze incomplete oxidation of a wide range of organic compounds by oxidative fermentation. Because the respiratory chain is a primary site of reactive oxygen species (ROS) production, the bacterium is expected to have a high capacity to detoxify nascent ROS. In the present study, a gene that encodes a catalase of G. oxydans, which might act as a potential scavenger of H(2)O(2), was cloned, and the expression product (termed rGoxCat) was characterized biochemically. rGoxCat is a heme b-containing tetrameric protein (molecular mass, 320 kDa) consisting of identical subunits. The recombinant enzyme displayed a strong catalase activity with a k(cat) of 6.28×10(4) s(-1) and a K(m) for H(2)O(2) of 61 mM; however, rGoxCat exhibited no peroxidase activity. These results, along with the phylogenetic position of the enzyme, provide conclusive evidence that rGoxCat is a monofunctional, large-subunit catalase. The enzyme was most stable in the pH range of 4-9, and greater than 60% of the original activity was retained after treatment at pH 3.0 and 40°C for 1h. Moreover, the enzyme exhibited excellent thermostability for a catalase from a mesophilic organism, retaining full activity after incubation for 30 min at 70°C. The observed catalytic properties of rGoxCat, as well as its stability in a slightly acidic environment, are consistent with its role in the elimination of nascent H(2)O(2) in a bacterium that produces a large amount of organic acid via oxidative fermentation.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Keigo
authors.initials: K
authors.lastname: Sugiyama
authors.forename: Miho
authors.initials: M
authors.lastname: Hosoya
authors.forename: Seiji
authors.initials: S
authors.lastname: Takahashi
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002374:Catalase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D020600:Gluconobacter oxydans / Q000201:enzymology / Q000235:genetics*; D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006418:Heme; D006861:Hydrogen Peroxide; D002374:Catalase