title: Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.
abstract: Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed.
authors.affiliation: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico, D.F., Mexico.
authors.forename: Martin
authors.initials: M
authors.lastname: Sará-Páez
authors.forename: Martha
authors.initials: M
authors.lastname: Contreras-Zentella
authors.forename: Saúl
authors.initials: S
authors.lastname: Gómez-Manzo
authors.forename: Alejandra Abigail
authors.initials: AA
authors.lastname: González-Valdez
authors.forename: Rolando
authors.initials: R
authors.lastname: Gasca-Licea
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mendoza-Hernández
authors.forename: José Edgardo
authors.initials: JE
authors.lastname: Escamilla
authors.forename: Horacio
authors.initials: H
authors.lastname: Reyes-Vivas
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D004579:Electron Transport; D041821:Gluconacetobacter / Q000201:enzymology*; D005948:Glucose Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone)