Functional and Biochemical Analysis of Glucose-6-Phosphate Dehydrogenase (G6PD) Variants: Elucidating the Molecular Basis of G6PD Deficiency

Abstract

G6PD deficiency is the most common enzymopathy, leading to alterations in the first step of the pentose phosphate pathway, which interferes with the protection of the erythrocyte against oxidative stress and causes a wide range of clinical symptoms of which hemolysis is one of the most severe. The G6PD deficiency causes several abnormalities that range from asymptomatic individuals to more severe manifestations that can lead to death. Nowadays, only 9.2% of all recognized variants have been related to clinical manifestations. It is important to understand the molecular basis of G6PD deficiency to understand how gene mutations can impact structure, stability, and enzymatic function. In this work, we reviewed and compared the functional and structural data generated through the characterization of 20 G6PD variants using different approaches. These studies showed that severe clinical manifestations of G6PD deficiency were related to mutations that affected the catalytic and structural nicotinamide adenine dinucleotide phosphate (NADPH) binding sites, and suggests that the misfolding or instability of the 3D structure of the protein could compromise the half-life of the protein in the erythrocyte and its activity.

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis

“…Finally, it is important to mention that although G6PD San Luis Potosi has been classified as a Class II mutant according to patients' hematological parameters, the recombinant G6PD variant showed a loss of catalysis similar to the previous values obtained for Class I mutants. The steady-state kinetic parameters obtained for the single mutant Class I G6PD Guadalajara and double mutant Class I G6PD Mount Sinai are similar to those reported for variants such as G6PDs Volendam, Andalus, Nashville, Durham, and Zacatecas, which lost nearly 90% of the catalytic constant (k cat ) with respect to WT GPD enzyme, which could explain the lack of reduced form of NADPH and counteract the oxidative stress on red blood cells, hence resulting in the most severe manifestation of the disease, chronic nonspherocytic hemolytic anemia [20][21][22]. The reclassification of the G6PD San Luis Potosi variant could allow a targeted treatment to the clinical hemolytic episodes of the patients with this G6PD mutation (in particular on malaria treatment), provide more information of the hemolytic risk and explain the severe clinical phenotypes by biochemical alterations on G6PD protein of these individuals.…”

“…The four clinical variants, three single mutants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and one double mutant (G6PD Mount Sinai), were generated by site-directed mutagenesis as previously described [19,21]. Polymerase chain reaction (PCR) was performed in a thermocycler (Mastercycler Eppendorf) using the plasmid pET-HisTEVP-g6pd containing the full human g6pd gene (NM_001042351 access) as a template.…”

Molecular Characterization of G6PD Mutations Reveals the High Frequency of G6PD Aures in the Mon-Khmer Population

“…Previous studies showed that the severe G6PD de ciency was caused by nucleotide mutation at or close to the NADP + or G6P binding site (3,35), which promoted the structural instability of G6PD and resulted in less NADPH production and oxidative stress sensitivity. The G6PD Aures mutation is commonly detected in Mediterranean populations, such as the Saudi Arabian (11.2-20%) (36)(37)(38), United Arab Emirates (11.9%) (39) and Kuwaiti (3.73%) (40).…”

“…The WHO has classi ed the G6PD Aures mutation as a class III mutation (43). The G6PD Aures was located between the sites of G6PD Vietmam-1 and Vietmam-2/Bahia, near G6PD Rignano, which was distant from the protein domain for NADP-1 binding (3,35). The mutation was predicted to affect the mini-instability of protein domain structure for binding with NADP-1 and to subsequently cause mild G6PD de ciency.…”

Laboratory diagnosis of G6PD deficiency. A British Society for Haematology Guideline

“…DNA sequence analysis has shown that the vast majority of mutations arise from single amino acid substitutions, mostly leading to a decrease in enzyme stability or to reduced catalytic efficiency. The degrees of enzymatic dysfunction detected in variants have been found to be in accordance with the severity of the clinical manifestations (Vulliamy et al, 1998;G omez-Manzo et al, 2017). A list of G6PD variants can be found at http://www.bioinf.org.uk/ g6pd/db.…”