title: Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan.
abstract: Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.
authors.affiliation: Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, México D.F. 04530, Mexico. saulmanzo@ciencias.unam.mx.
authors.forename: Saúl
authors.initials: S
authors.lastname: Gómez-Manzo
authors.affiliation: CONACYT-Instituto Nacional de Pediatría, México D.F. 04530, Mexico. jmarcialqu@conacyt.mx.
authors.forename: Jaime
authors.initials: J
authors.lastname: Marcial-Quino
authors.affiliation: Laboratorio de Neurociencias, Instituto Nacional de Pediatría, México D.F. 04530, Mexico. america_vc@yahoo.com.mx.
authors.forename: America
authors.initials: A
authors.lastname: Vanoye-Carlo
authors.affiliation: CONACYT-Laboratorio de Bioingeniería, Universidad de Colima, Colima 28400, Mexico. hjserranopo@conacyt.mx.
authors.forename: Hugo
authors.initials: H
authors.lastname: Serrano-Posada
authors.affiliation: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico. abigaila@correo.biomedicas.unam.mx.
authors.forename: Abigail
authors.initials: A
authors.lastname: González-Valdez
authors.affiliation: Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, México D.F. 04530, Mexico. razer_45r@hotmail.com.
authors.forename: Víctor
authors.initials: V
authors.lastname: Martínez-Rosas
authors.affiliation: Laboratorio de Inmunoquímica, Hospital Infantil de México Federico Gómez, México D.F. 06720, Mexico. beatrizhb_16@comunidad.unam.mx.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Hernández-Ochoa
authors.affiliation: Colegio de Ciencias y Humanidades, Plantel Casa Libertad, UACM, México D.F. 09620, Mexico. edgar.sierra@uacm.edu.mx.
authors.forename: Edgar
authors.initials: E
authors.lastname: Sierra-Palacios
authors.affiliation: CONACYT-Instituto Nacional de Pediatría, México D.F. 04530, Mexico. racastilloro@conacyt.mx.
authors.forename: Rosa Angélica
authors.initials: RA
authors.lastname: Castillo-Rodríguez
authors.affiliation: Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, México D.F. 04510, Mexico. miguel.ccqi@yahoo.com.mx.
authors.forename: Miguel
authors.initials: M
authors.lastname: Cuevas-Cruz
authors.affiliation: Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, México D.F. 04510, Mexico. e-rodriguez-bustamante@ciencias.unam.mx.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Rodríguez-Bustamante
authors.affiliation: Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, México D.F. 04510, Mexico. arrespin@unam.mx.
authors.forename: Roberto
authors.initials: R
authors.lastname: Arreguin-Espinosa
mesh_terms: D044467:American Indian or Alaska Native / Q000235:genetics*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D018360:Crystallography, X-Ray; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008800:Mexico; D008958:Models, Molecular; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase
keywords: glucose-6-phosphate dehydrogenase (G6PD) deficiency; human G6PD mutants; steady state kinetics; structural characterization; thermostability