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The stability of G6PD is affected by mutations with different clinical phenotypes.

Gómez-Manzo S, Terrón-Hernández J, De la Mora-De la Mora I, González-Valdez A, Marcial-Quino J, García-Torres I, Vanoye-Carlo A, López-Velázquez G, Hernández-Alcántara G, Oria-Hernández J, Reyes-Vivas H, Enríquez-Flores S - Int J Mol Sci (2014)

Bottom Line: For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes.This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile.The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, México D.F. 04530, Mexico. saulmanzo@ciencias.unam.mx.

ABSTRACT
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The k(cat) (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.

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Thermal stability of human G6PD enzymes. (A) Thermal unfolding of WT G6PD and the mutants (0.8 mg/mL) in 25 mM NaPO4 pH 7.4 was monitored by recording the change in CD signal at 222 nm at different temperatures ranging from 20 to 90 °C. The unfolded fraction of protein and the Tm (melting temperature midpoint of the transition values) (inset) were calculated as previously reported [22]; and (B) Thermal inactivation assays of WT G6PD and the mutants after incubation for 20 min at the indicated temperature. The T50 (temperature where 50% of its original activity is retained) after incubation at different temperatures for 20 min is shown. In all cases, 200 ng of total protein was used. The assays were performed in duplicate; standard errors were lower than 5%.
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ijms-15-21179-f004: Thermal stability of human G6PD enzymes. (A) Thermal unfolding of WT G6PD and the mutants (0.8 mg/mL) in 25 mM NaPO4 pH 7.4 was monitored by recording the change in CD signal at 222 nm at different temperatures ranging from 20 to 90 °C. The unfolded fraction of protein and the Tm (melting temperature midpoint of the transition values) (inset) were calculated as previously reported [22]; and (B) Thermal inactivation assays of WT G6PD and the mutants after incubation for 20 min at the indicated temperature. The T50 (temperature where 50% of its original activity is retained) after incubation at different temperatures for 20 min is shown. In all cases, 200 ng of total protein was used. The assays were performed in duplicate; standard errors were lower than 5%.

Mentions: Protein stability has been extensively studied in human G6PD, and it has already been demonstrated that several mutations can induce G6PD deficiency by decreasing its stability [7,18,19,20]. To assess whether the diminished catalytic activity of the studied mutant enzymes was due to an alteration in protein stability, we evaluated the thermal denaturation of the four G6PD enzymes. The global stability of the proteins was followed as the change in the CD signal at 222 nm. The results indicate a two-state process with a Tm (melting temperature midpoint of the transition) of 54.3 °C for the WT G6PD enzyme (Figure 4A). This result differs marginally from the recently published Tm value of 51.5 °C obtained for recombinant G6PD-HisTEVP [17]. However, it is consistent with a previously reported Tm value for the WT G6PD enzyme of approximately 55 °C [14]. The Tm was 54.3 °C for WT G6PD and 53 °C for the Yucatan, Valladolid and Mexico City G6PDs. This change in the Tm was not significant; however, the structural stability of the Nashville G6PD enzyme shows a strong difference because the Tm was 50 °C, 5 °C below the value obtained for the WT G6PD enzyme, indicating that this enzyme is more sensitive to temperature denaturation.


The stability of G6PD is affected by mutations with different clinical phenotypes.

Gómez-Manzo S, Terrón-Hernández J, De la Mora-De la Mora I, González-Valdez A, Marcial-Quino J, García-Torres I, Vanoye-Carlo A, López-Velázquez G, Hernández-Alcántara G, Oria-Hernández J, Reyes-Vivas H, Enríquez-Flores S - Int J Mol Sci (2014)

Thermal stability of human G6PD enzymes. (A) Thermal unfolding of WT G6PD and the mutants (0.8 mg/mL) in 25 mM NaPO4 pH 7.4 was monitored by recording the change in CD signal at 222 nm at different temperatures ranging from 20 to 90 °C. The unfolded fraction of protein and the Tm (melting temperature midpoint of the transition values) (inset) were calculated as previously reported [22]; and (B) Thermal inactivation assays of WT G6PD and the mutants after incubation for 20 min at the indicated temperature. The T50 (temperature where 50% of its original activity is retained) after incubation at different temperatures for 20 min is shown. In all cases, 200 ng of total protein was used. The assays were performed in duplicate; standard errors were lower than 5%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264219&req=5

ijms-15-21179-f004: Thermal stability of human G6PD enzymes. (A) Thermal unfolding of WT G6PD and the mutants (0.8 mg/mL) in 25 mM NaPO4 pH 7.4 was monitored by recording the change in CD signal at 222 nm at different temperatures ranging from 20 to 90 °C. The unfolded fraction of protein and the Tm (melting temperature midpoint of the transition values) (inset) were calculated as previously reported [22]; and (B) Thermal inactivation assays of WT G6PD and the mutants after incubation for 20 min at the indicated temperature. The T50 (temperature where 50% of its original activity is retained) after incubation at different temperatures for 20 min is shown. In all cases, 200 ng of total protein was used. The assays were performed in duplicate; standard errors were lower than 5%.
Mentions: Protein stability has been extensively studied in human G6PD, and it has already been demonstrated that several mutations can induce G6PD deficiency by decreasing its stability [7,18,19,20]. To assess whether the diminished catalytic activity of the studied mutant enzymes was due to an alteration in protein stability, we evaluated the thermal denaturation of the four G6PD enzymes. The global stability of the proteins was followed as the change in the CD signal at 222 nm. The results indicate a two-state process with a Tm (melting temperature midpoint of the transition) of 54.3 °C for the WT G6PD enzyme (Figure 4A). This result differs marginally from the recently published Tm value of 51.5 °C obtained for recombinant G6PD-HisTEVP [17]. However, it is consistent with a previously reported Tm value for the WT G6PD enzyme of approximately 55 °C [14]. The Tm was 54.3 °C for WT G6PD and 53 °C for the Yucatan, Valladolid and Mexico City G6PDs. This change in the Tm was not significant; however, the structural stability of the Nashville G6PD enzyme shows a strong difference because the Tm was 50 °C, 5 °C below the value obtained for the WT G6PD enzyme, indicating that this enzyme is more sensitive to temperature denaturation.

Bottom Line: For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes.This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile.The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, México D.F. 04530, Mexico. saulmanzo@ciencias.unam.mx.

ABSTRACT
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The k(cat) (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.

Show MeSH
Related in: MedlinePlus