Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. Furthermore, for G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. Here, we characterized the functional and structural properties of nine G6PD variants: G6PD Gaohe, G6PD Mahidol, G6PD Shoklo, G6PD Canton, G6PD Kaiping, G6PD Gaohe + Kaiping, G6PD Mahidol + Canton, G6PD Mahidol + Kaiping and G6PD Canton + Kaiping. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. The combined effects of two mutations were observed, with the Canton mutation reducing structural stability and the Kaiping mutation increasing it in the double mutations. Severe enzyme deficiency in the double mutants was mainly determined by the trade-off between protein stability and catalytic activity. Additionally, it was demonstrated that AG1, a G6PD activator, only marginally increased G6PD enzymatic activity and stability.

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure

“…Multiple mutations were previously shown to have a greater effect on G6PD deficiency than single mutations ( Nantakomol et al, 2013 ). The combined effects of two or more mutations on catalytic activity and structural stability were observed ( Praoparotai et al, 2020 ; Pakparnich et al, 2021 ). Here, G6PD Songklanagarind + Viangchan and G6PD Chinese 4 + Viangchan variants were observed in female individuals with extremely low enzyme activity.…”

“…Primers used for site-directed mutation are listed in Table 3 . The PCR conditions for site-directed mutagenesis were previously described ( Pakparnich et al, 2021 ). G6PD protein expression was performed using E. coli BL21 (DE3).…”

Molecular dynamics of G6PD variants from sub-Saharan Africa

“…This supports the conjecture of allosteric impact of the AMIN variant and suggests future work to include longer simulation times, and detailed assessments of allosteric modulation. Recent work has shown that AG1 only has marginal effects on a diverse set of G6PD variants (Mahidol, Kaiping, Gaohe, Canton, Shoklo and combinations of these variants) [33] . This reinforces the need to investigate such molecules in diverse populations.…”

Cytochrome P450 2D6 (CYP2D6) and glucose-6-phosphate dehydrogenase (G6PD) genetic variations in Thai vivax malaria patients: Implications for 8-aminoquinoline radical cure

“…Primers used for site-directed mutation are listed in S1 Table . The PCR conditions for site-directed mutagenesis were previously described [ 40 ]. G6PD protein expression was achieved using E .…”

“…G6PD mutations were created by site-directed mutagenesis using pET28a-G6PD WT as a template and the presence of desired mutations was confirmed by DNA sequencing. Primers used for site-directed mutation are listed in S1 Table . The PCR conditions for site-directed mutagenesis were previously described [40]. G6PD protein expression was achieved using E. coli BL21 (DE3), which were cultured at 37˚C with 250 rpm shaking in the presence of 50 μg/mL kanamycin until the OD 600 reached 1 and G6PD expression was induced using 1 mM IPTG.…”