Marked decrease in specific activity contributes to disease phenotype in two human glucose 6-phosphate dehydrogenase mutants, G6PD_Unionand G6PD_Andalus

Abstract

Clones overexpressing clinical glucose 6-phosphate dehydrogenase (G6PD) mutants Union (c.1360C>T / p.Arg454Cys) and Andalus (c.1361G>A / p.Arg454His) , have been constructed. These abolish a salt bridge between Arg454 and Asp 286. One mutant is reportedly a Class II clinical variant and the other a Class I. Kinetic studies of the purified proteins reveal that, for both mutants, k cat is about 10-fold decreased, thus giving a 90% decrease in the WHO assay, and also presumably under physiological conditions. In contrast with unfavourable changes in V max for both mutants, K m values for both G6P and NADP + are decreased approximately 5-fold. Measurements with alternative substrates confirm that G6PD Union, like the wild-type enzyme, follows a rapid-equilibrium random-order mechanism, allowing calculation of enzyme-substrate dissociation constants from initial-rate parameters. The mutations result in several-fold tighter binding of glucose 6-phosphate to the free enzyme. Binding, however, is clearly less productive than with normal enzyme. G6PD mutations are thought to cause haemolytic anaemia by compromising enzyme stability. Both these mutants indeed show somewhat decreased thermostability. However, at 37 o C and with NADP + , the stability differences are only moderate. Decreased catalytic efficiency clearly contributes to the disease phenotype of these two mutants, entirely accounting for reported decrease in leukocyte G6PD levels, though not for still lower levels in erythrocytes. Neither the kinetic nor the stability effects appear to justify the different clinical classification of these mutations.

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

“…Several systems have been implemented to achieve expression and purification of both the wild-type (WT) and G6PD variants. Heterologous expression of human G6PD based on Escherichia coli strains and compatible plasmids have been widely used to produce high protein quantities for structural and functional studies [12,14,16,17,[19][20][21][23][24][25][26]. During the first characterization studies, the G6PD gene was widely fused to different expression vectors such as pKK223-3, pKK233-2, pMPM-A4Ω and pPLcmu299 [15].…”

“…These results revealed that this new system was very useful for the expression of normal and mutant enzymes. Subsequently, pTrc99A [16,18,25] was chosen as the expression vector; however, it was not compatible with commercial E. coli strains.…”

“…At low temperatures, the recombinant protein can reach a proper folding. Several groups have performed the expression of both WT and mutants of G6PD at different temperatures from 15 to 37 • C [14][15][16][17][18][19][20][21]24,25]. Specifically, Gómez-Manzo et al [19][20][21], found that soluble G6PD levels were highest at 25 • C for the G6PD Yucatan, Valladolid, Mexico City, Nashville, Durham, Santa-Maria, A+, Zacatecas, Viangchan and Vanua-Lava variants.…”

“…On the other hand, Boonyuen et al [22] expressed G6PD Mahidol and Viangchan variants at 20 • C, inducing with 1 mM IPTG at 20 h, while the G6PD Plymouth and Mahidol were expressed at 37 • C for 24 h IPTG induction [18]. Furthermore, the G6PD Unión and Andalus variants were grown at 37 • C [16] and the clinical Wisconsin and Nashville variants were allowed to grow for another 5 h at 30 • C. Finally, the G6PD Volendam expression was obtained using 2% of arabinose as an inducer [15]. The vector selection and optimization of the culture conditions are crucial steps for the optimal expression level of G6PD.…”

Glucosa 6 fosfato deshidrogenasa en indígenas piaroas en zona endémica de malaria.

“…Ahora bien, si la mezcla génica fuera efectiva entre indígenas Piaroas e individuos de las ciudad (que la población indígena este integrada a la mestiza urbana y no aislada o desplazada), es de esperar deficiencia bioquímica de G6PD clase III, la que caracteriza a la población venezolana y que se corresponde con individuos con deficiencia enzimática de leve a moderada, en los cuales la sintomatología hemolítica es desencadenada únicamente por la exposición a sustancias que inducen estrés oxidante 18,39,40 .…”