Hernández-Ochoa Beatriz, Gualos-González Mónica Guadalupe, Moreno-Hernández Jhuremy Alexandra, Morales-Luna Laura, Vázquez-Bautista Montserrat, Canseco-Ávila Luis Miguel, Pérez de la Cruz Verónica, Arreguin-Espinosa Roberto, Hernández-Urzua Elizabeth, Enríquez-Flores Sergio, De la Mora-De la Mora Ignacio, Cárdenas-Rodríguez Noemí, Bandala Cindy, De Franceschi Lucia, Vidal-Limon Abraham, Gómez-Manzo Saúl
Laboratorio de Inmunoquímica, Hospital Infantil de México Federico Gómez, Secretaría de Salud, Mexico City 06720, Mexico.
Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City 04530, Mexico.
Int J Mol Sci. 2025 Aug 30;26(17):8464. doi: 10.3390/ijms26178464.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread enzymopathy affecting approximately 500 million individuals that represents a significant global health issue. Among the more than 230 identified mutations in the G6PD gene, six class A variants-G6PD Utrecht (Pro409Ser), G6PD Suwalki (Pro409Arg), G6PD Merlo (Pro409Gln), G6PD Kawasaki (Gly410Ala), G6PD Shinagawa (Gly410Asp), and G6PD Riverside (Gly410Cys)-are located in the beta-loop near the NADP binding site. These mutations are of particular interest due to their association with severe hematologic phenotypes, including chronic hemolytic anemia, as well as their proposed role in the allosteric regulation of G6PD multimerization. This study presents a comprehensive biochemical and functional characterization of these clinically relevant G6PD variants. The variant enzymes were cloned, expressed, and purified for characterization. Kinetic parameters and thermal stability assays, complemented by molecular dynamics simulations (MDS), were employed to elucidate the structural impacts of the mutations. Our results demonstrate that these mutations significantly impair protein function, characterized by reduced affinity for glucose-6-phosphate (G6P) and NADP, as well as altered thermal stability compared with wild-type G6PD. MDS revealed that point mutations in the βN- and βM-sheets in the NADP region propagate subtle conformational changes, ultimately affecting the NADPc region and the G6P binding cavity. Furthermore, secondary structure element analyses of the simulation data showed that Pro409 and Gly410 point mutations propagate several changes around residues 195-210 (G6P binding site) and 380-400 (NADP), explaining their effect on overall catalytic performance. These findings enhance our understanding of the molecular mechanisms underlying G6PD deficiency and its clinical implications, providing a foundation for future therapeutic strategies aimed at mitigating the effects of these variants.
葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是一种广泛存在的酶病，影响着约5亿人，是一个重大的全球健康问题。在G6PD基因中已鉴定出的230多种突变中，六种A类变体——G6PD乌得勒支（Pro409Ser）、G6PD苏瓦尔基（Pro409Arg）、G6PD梅洛（Pro409Gln）、G6PD川崎（Gly410Ala）、G6PD品川（Gly410Asp）和G6PD河滨（Gly410Cys）——位于NADP结合位点附近的β环中。这些突变特别令人关注，因为它们与严重的血液学表型有关，包括慢性溶血性贫血，以及它们在G6PD多聚化的变构调节中所起的作用。本研究对这些临床相关的G6PD变体进行了全面的生化和功能表征。将变体酶进行克隆、表达和纯化以进行表征。采用动力学参数和热稳定性测定，并辅以分子动力学模拟（MDS），以阐明突变的结构影响。我们的结果表明，与野生型G6PD相比，这些突变显著损害蛋白质功能，其特征是对葡萄糖-6-磷酸（G6P）和NADP的亲和力降低，以及热稳定性改变。MDS显示，NADP区域βN和βM片层中的点突变传播了细微的构象变化，最终影响NADPc区域和G6P结合腔。此外，对模拟数据的二级结构元件分析表明，Pro409和Gly410点突变在残基195 - 210（G6P结合位点）和380 - 400（NADP）周围传播了多种变化，解释了它们对整体催化性能的影响。这些发现加深了我们对G6PD缺乏症潜在分子机制及其临床意义的理解，为未来旨在减轻这些变体影响的治疗策略奠定了基础。
