Abstract
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.
Highlights
Gluconacetobacter diazotrophicus PAL5 (GDI) is a strict aerobe and a nitrogen-fixing acetic acid bacterium that was originally isolated from sugar cane [1]
The cloned zwf sequence analyzed by blast nucleotide (BLASTn) sequencing in GenBank found a 100% similarity with the nucleotide sequence of glucose-6-phosphate dehydrogenase (G6PD) from GDI (GenBank: CAP54231.1) [13]
A comparison of the G. diazotrophicus PAL5 G6PD amino acid sequence was performed with HHblits [14] and revealed 100% levels of similarity with the putative G6PD from Tanticharoenia sakaeratensis, Verrucomicrobiae bacterium, and Xanthomonas cucurbitae, among others (Table S1)
Summary
Gluconacetobacter diazotrophicus PAL5 (GDI) is a strict aerobe and a nitrogen-fixing acetic acid bacterium that was originally isolated from sugar cane [1]. It grows under stringent conditions, such as in areas with a high sucrose content and low pH [2]. Whole genome sequencing allows for better understanding of the GDI, and the knowledge of several molecular and biochemical aspects of this microorganism have opened new research directions. The GDI genome is composed of one chromosome (3.9 Mb) and two plasmids (16.6 and 38.8 kb, respectively). About 3938 coding sequences have been annotated, including those related to sugar metabolism, such as that of the pentose phosphate pathway (PPP) [7,8]
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