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Molecular Cloning and Exploration of the Biochemical and Functional Analysis of Recombinant Glucose-6-Phosphate Dehydrogenase from Gluconoacetobacter diazotrophicus PAL5

1
Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
2
Laboratorio de Nutrición Experimental, Instituto Nacional de Pediatría, Secretaría de Salud 04530, Mexico
3
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de Mexico 04510, Mexico
4
Consejo Nacional de Ciencia y Tecnología (CONACYT), Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de Mexico 04530, Mexico
5
Institute for Bio- and Geosciences (IBG-2: Plant Sciences), Forschungszentrum Jülich, Wilhelm Johnen Straße, 52428 Jülich, Germany
6
Laboratorio de Inmunoquímica, Hospital Infantil de México Federico Gómez, Secretaría de Salud, Ciudad de Mexico 06720, Mexico
7
Laboratorio de Neurociencias, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
8
Consejo Nacional de Ciencia y Tecnología (CONACYT), Laboratorio de Agrobiotecnología, Tecnoparque CLQ, Universidad de Colima, Carretera los Limones-Loma de Juárez, Colima 28629, Mexico
9
Colegio de Ciencias y Humanidades, Plantel Casa Libertad, Universidad Autónoma de la Ciudad de México, Ciudad de Mexico 09620, Mexico
10
Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de Mexico, Ciudad de Mexico 04510, Mexico
11
Departamento de Infectología, Hospital Infantil de México Federico Gómez, Dr. Márquez No. 162, Col Doctores, Delegación Cuauhtémoc 06720, Mexico
12
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, S.S.A., Ciudad de Mexico 14269, Mexico
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(21), 5279; https://doi.org/10.3390/ijms20215279
Received: 17 September 2019 / Revised: 21 October 2019 / Accepted: 22 October 2019 / Published: 24 October 2019
(This article belongs to the Special Issue Computational Studies of Biomolecules)
Gluconacetobacter diazotrophicus PAL5 (GDI) is an endophytic bacterium with potential biotechnological applications in industry and agronomy. The recent description of its complete genome and its principal metabolic enzymes suggests that glucose metabolism is accomplished through the pentose phosphate pathway (PPP); however, the enzymes participating in this pathway have not yet been characterized in detail. The objective of the present work was to clone, purify, and biochemically and physicochemically characterize glucose-6-phosphate dehydrogenase (G6PD) from GDI. The gene was cloned and expressed as a tagged protein in E. coli to be purified by affinity chromatography. The native state of the G6PD protein in the solution was found to be a tetramer with optimal activity at pH 8.8 and a temperature between 37 and 50 °C. The apparent Km values for G6P and nicotinamide adenine dinucleotide phosphate (NADP+) were 63 and 7.2 μM, respectively. Finally, from the amino acid sequence a three-dimensional (3D) model was obtained, which allowed the arrangement of the amino acids involved in the catalytic activity, which are conserved (RIDHYLGKE, GxGGDLT, and EKPxG) with those of other species, to be identified. This characterization of the enzyme could help to identify new environmental conditions for the knowledge of the plant–microorganism interactions and a better use of GDI in new technological applications. View Full-Text
Keywords: glucose 6 phosphate dehydrogenase (G6PD); bioinformatics analysis; kinetic parameters; thermal stability; three-dimensional structure glucose 6 phosphate dehydrogenase (G6PD); bioinformatics analysis; kinetic parameters; thermal stability; three-dimensional structure
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Ramírez-Nava, E.J.; Ortega-Cuellar, D.; González-Valdez, A.; Castillo-Rodríguez, R.A.; Ponce-Soto, G.Y.; Hernández-Ochoa, B.; Cárdenas-Rodríguez, N.; Martínez-Rosas, V.; Morales-Luna, L.; Serrano-Posada, H.; Sierra-Palacios, E.; Arreguin-Espinosa, R.; Cuevas-Cruz, M.; Rocha-Ramírez, L.M.; Pérez de la Cruz, V.; Marcial-Quino, J.; Gómez-Manzo, S. Molecular Cloning and Exploration of the Biochemical and Functional Analysis of Recombinant Glucose-6-Phosphate Dehydrogenase from Gluconoacetobacter diazotrophicus PAL5. Int. J. Mol. Sci. 2019, 20, 5279.

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