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Purification and Characterization of the Membrane-Bound Quinoprotein Glucose Dehydrogenase of Gluconacetobacter diazotrophicus PAL 5

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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.

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Abbreviations

ATCC:

American type culture collection

Cyt-a1:

Cytochrome a1

DMSO:

Dimethyl sulfoxide

FAD:

Flavin adenine dinucleotide

KP-T:

Phosphate buffer plus 0.1 % Triton X-100

NTB:

Nitro blue tetrazolium

Q7:

Quinone seven isoprene

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Acknowledgments

This work is a requisite to obtain the PhD grade in Science; we appreciate to Posgrado en Ciencias Biológicas UNAM, for the academic training of Martín Sará Páez during its doctorate studies. We also appreciate to Dirección General de Estudios de Posgrado (DGEP), for the scholarship granted to MS-P during his studies. SG-M is supported by Consejo Nacional de Ciencia y Tecnología Grant 154570. The technical assistance of Javier Gallegos Infante (IFC/UNAM) for assistance in bibliographic materials is greatly appreciated. This work was started by the Prof. José Edgardo Escamilla in the Instituto de Fisiología Célular, UNAM; but unfortunately deceased, and was concluded by the Dr. Horacio Reyes Vivas from Instituto Nacional de Pediatría. The authors concluded this project that now is published in his honor and memory.

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Authors and Affiliations

  1. Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico, D.F., Mexico

    Martin Sará-Páez, Martha Contreras-Zentella, Rolando Gasca-Licea & José Edgardo Escamilla

  2. Laboratorio de Bioquímica-Genética, Torre de Investigación, Instituto Nacional de Pediatría, Secretaría de Salud, Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, D.F., Mexico

    Martin Sará-Páez, Saúl Gómez-Manzo & Horacio Reyes-Vivas

  3. Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico, D.F., Mexico

    Alejandra Abigail González-Valdez

  4. Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, D.F., Mexico

    Guillermo Mendoza-Hernández

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  1. Martin Sará-Páez
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  2. Martha Contreras-Zentella
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  7. José Edgardo Escamilla
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  8. Horacio Reyes-Vivas
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Correspondence to Horacio Reyes-Vivas.

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Sará-Páez, M., Contreras-Zentella, M., Gómez-Manzo, S. et al. Purification and Characterization of the Membrane-Bound Quinoprotein Glucose Dehydrogenase of Gluconacetobacter diazotrophicus PAL 5. Protein J 34, 48–59 (2015). https://doi.org/10.1007/s10930-014-9596-4

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