Evaluation of Three Mutations in Codon 385 of Glucose-6-Phosphate Dehydrogenase via Biochemical and In Silico Analysis

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an enzymopathy that affects approximately 500 million people worldwide. A great number of mutations in the gene have been described. However, three class A G6PD variants known as G6PD Tomah (C385R), G6PD Kangnam (C385G), and G6PD Madrid (C385W) have been reported to be clinically important due to their associations with severe clinical manifestations such as hemolytic anemia. Therefore, this work aimed to perform, for the first time, biochemical and functional characterizations of these variants. The G6PD variants were cloned and purified for this purpose, followed by analyses of their kinetic parameters and thermal stability, as well as in silico studies. The results showed that the mutations induced changes in the proteins. Regarding the kinetic parameters, it was observed that the three variants showed lower affinities for G6P and NADP , as well as lower thermal stability compared to WT-G6PD. Molecular dynamics simulations showed that C385 mutations induced changes around neighboring amino acids. Metadynamics simulations showed that most remarkable changes account for the binding pocket volumes, particularly in the structural NADP binding site, with a concomitant loss of affinity for catalytic processes.