{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T21:22:40Z","timestamp":1762809760179,"version":"build-2065373602"},"reference-count":63,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,8,30]],"date-time":"2025-08-30T00:00:00Z","timestamp":1756512000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Pediatrics, Mexico City, Mexico","award":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"],"award-info":[{"award-number":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"]}]},{"name":"INP","award":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"],"award-info":[{"award-number":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"]}]},{"name":"Fondos Federales","award":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"],"award-info":[{"award-number":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"]}]},{"name":"Consejo Nacional de Humanidades, Ciencias y Tecnolog\u00edas (CONAHCYT) Ciencia de Frontera 2023","award":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"],"award-info":[{"award-number":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"]}]},{"name":"National Supercomputing Center","award":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"],"award-info":[{"award-number":["034\/2022","HIM\/2019\/036 SSA. 1595","CF-2023-I-811","TKII-AMVL001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2014G6PD Utrecht (Pro409Ser), G6PD Suwalki (Pro409Arg), G6PD Merlo (Pro409Gln), G6PD Kawasaki (Gly410Ala), G6PD Shinagawa (Gly410Asp), and G6PD Riverside (Gly410Cys)\u2014are 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 \u03b2N- and \u03b2M-sheets in the NADP+s region propagate subtle conformational changes, ultimately affecting the NADP+c 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\u2013210 (G6P binding site) and 380\u2013400 (NADP+s), 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.<\/jats:p>","DOI":"10.3390\/ijms26178464","type":"journal-article","created":{"date-parts":[[2025,9,2]],"date-time":"2025-09-02T15:02:57Z","timestamp":1756825377000},"page":"8464","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Integrated Approach for Biochemical and Functional Characterization of Six Clinical Variants of Glucose-6-Phosphate Dehydrogenase"],"prefix":"10.3390","volume":"26","author":[{"given":"Beatriz","family":"Hern\u00e1ndez-Ochoa","sequence":"first","affiliation":[{"name":"Laboratorio de Inmunoqu\u00edmica, Hospital Infantil de M\u00e9xico Federico G\u00f3mez, Secretar\u00eda de Salud, Mexico City 06720, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M\u00f3nica Guadalupe","family":"Gualos-Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica Gen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jhuremy Alexandra","family":"Moreno-Hern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica Gen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7194-4522","authenticated-orcid":false,"given":"Laura","family":"Morales-Luna","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica Gen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"},{"name":"Posgrado en Ciencias Biol\u00f3gicas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Mexico City 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4651-7892","authenticated-orcid":false,"given":"Montserrat","family":"V\u00e1zquez-Bautista","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica Gen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"},{"name":"Programa de Posgrado en Biomedicina y Biotecnolog\u00eda Molecular, Escuela Nacional de Ciencias Biol\u00f3gicas, Instituto Polit\u00e9cnico Nacional, Mexico City 11340, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5694-3118","authenticated-orcid":false,"given":"Luis Miguel","family":"Canseco-\u00c1vila","sequence":"additional","affiliation":[{"name":"Laboratorio de Diagn\u00f3stico y Biomedicina Molecular, Facultad de Ciencias Qu\u00edmicas, Campus IV, Universidad Aut\u00f3noma de Chiapas, Tapachula City 30580, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1451-8754","authenticated-orcid":false,"given":"Ver\u00f3nica","family":"P\u00e9rez de la Cruz","sequence":"additional","affiliation":[{"name":"Neurobiochemistry and Behavior Laboratory, National Institute of Neurology and Neurosurgery \u201cManuel Velasco Su\u00e1rez\u201d, Mexico City 14269, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4611-4770","authenticated-orcid":false,"given":"Roberto","family":"Arreguin-Espinosa","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica de Biomacromol\u00e9culas, Instituto de Qu\u00edmica, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Mexico City 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth","family":"Hern\u00e1ndez-Urzua","sequence":"additional","affiliation":[{"name":"Laboratorio de Toxicolog\u00eda Gen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2058-5707","authenticated-orcid":false,"given":"Sergio","family":"Enr\u00edquez-Flores","sequence":"additional","affiliation":[{"name":"Laboratorio de Biomol\u00e9culas y Salud Infantil, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5654-8089","authenticated-orcid":false,"given":"Ignacio","family":"De la Mora-De la Mora","sequence":"additional","affiliation":[{"name":"Laboratorio de Biomol\u00e9culas y Salud Infantil, Instituto Nacional de Pediatr\u00eda, 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(INECOL), Carretera Antigua a Coatepec 351, El Haya, Xalapa 91073, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2095-9182","authenticated-orcid":false,"given":"Sa\u00fal","family":"G\u00f3mez-Manzo","sequence":"additional","affiliation":[{"name":"Laboratorio de Bioqu\u00edmica Gen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Secretar\u00eda de Salud, Mexico City 04530, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"21179","DOI":"10.3390\/ijms151121179","article-title":"The Stability of G6PD Is Affected by Mutations with Different Clinical Phenotypes","volume":"15","year":"2014","journal-title":"Int. J. Mol. 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