Cloning and biochemical characterization of three glucose‑6‑phosphate dehydrogenase mutants presents in the Mexican population

The deficiency of glucose‑6‑phosphate dehydrogenase (G6PD) is one of the most common inborn errors of metabolism worldwide. This congenital disorder generally results from mutations that are spread throughout the entire gene of G6PD. Three single-point mutations for G6PD have been reported in the Mexican population and named Veracruz (Arg365His), G6PD Seattle (Asp282His), and G6PD Mexico DF (Thr65Ala), whose biochemical characterization have not yet been studied. For this reason, in this work we analyzed the putative role of the three mutations to uncover the functional consequences on G6PD activity. To this end, was developed a method to clone, overexpress, and purify recombinant human G6PD. The results obtained from all variants showed a loss of catalysis by 80 to 97% and had a decrease in affinity for both physiological substrates with respect to the wild type (WT) G6PD. Our results also showed that the three mutations affected three-dimensional structure and protein stability, suggesting an unstable structure with low conformational stability that affected its G6PD functionality. Finally, based on the biochemical characterization of the unclassified G6PD Mexico DF, we suggest that this variant could be grouped as a Class I variant, because biochemical data are similar with other Class I G6PDs. •Analysis of three variants of the G6PD human identified in the Mexican population•Construction of G6PD mutants (Veracruz, Seattle, Mexico DF) and their overexpression in E. coli•The kinetic parameters obtained of all variants showed a loss of catalysis with respect to the WT G6PD.•The mutations showed effects in the stability of the tertiary structure of the protein.•The 3D structure from each G6PD mutant allowed identifying changes in the structure of the protein.