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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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Related in: MedlinePlus

Structural characterization of human recombinant wild-type (WT) G6PD and mutants. Far-UV circular dichroism (CD) spectra of WT G6PD and mutants are shown. The protein concentration was 0.8 mg/mL in all cases. The experiments were performed in duplicate; standard errors were less than 5%.
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ijms-15-21179-f003: Structural characterization of human recombinant wild-type (WT) G6PD and mutants. Far-UV circular dichroism (CD) spectra of WT G6PD and mutants are shown. The protein concentration was 0.8 mg/mL in all cases. The experiments were performed in duplicate; standard errors were less than 5%.

Mentions: Because the G6PD variant enzymes have diminished catalytic efficiency, it was necessary to determine whether these conformational changes are the result of a wider structural disruption or a local effect. The secondary structures of the WT G6PD and the clinical variants were evaluated spectroscopically by circular dichroism (CD). As shown in Figure 3, the CD spectra of the mutant enzymes were comparable in pattern and intensity to the WT enzyme, indicating that the mutations did not significantly alter the secondary structure of the proteins.


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)

Structural characterization of human recombinant wild-type (WT) G6PD and mutants. Far-UV circular dichroism (CD) spectra of WT G6PD and mutants are shown. The protein concentration was 0.8 mg/mL in all cases. The experiments were performed in duplicate; standard errors were less than 5%.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-15-21179-f003: Structural characterization of human recombinant wild-type (WT) G6PD and mutants. Far-UV circular dichroism (CD) spectra of WT G6PD and mutants are shown. The protein concentration was 0.8 mg/mL in all cases. The experiments were performed in duplicate; standard errors were less than 5%.
Mentions: Because the G6PD variant enzymes have diminished catalytic efficiency, it was necessary to determine whether these conformational changes are the result of a wider structural disruption or a local effect. The secondary structures of the WT G6PD and the clinical variants were evaluated spectroscopically by circular dichroism (CD). As shown in Figure 3, the CD spectra of the mutant enzymes were comparable in pattern and intensity to the WT enzyme, indicating that the mutations did not significantly alter the secondary structure of the proteins.

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