title: Enlightening the molecular basis of trypanothione specificity in trypanosomatids: mutagenesis of Leishmania infantum glyoxalase II.
abstract: Leishmania infantum glyoxalase II shows absolute specificity towards its trypanothione thioester substrate. In the previous work, we performed a comparative analysis of glyoxalase II structures determined by X-ray crystallography which revealed that Tyr291 and Cys294, absent in the human homologue, are essential for substrate binding. To validate this trypanothione specificity hypothesis we produced a mutant L. infantum GLO2 enzyme by replacing Tyr291 and Cys294 by arginine and lysine, respectively. This new enzyme is capable to use the glutathione thioester substrate, with kinetic parameters similar to the ones from the human enzyme. Substrate specificity is likely to be mediated by spermidine moiety binding, providing a primer for understanding the molecular basis of trypanothione specificity.
authors.affiliation: Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Ed. C8, Campo Grande, 1749-016 Lisboa, Portugal.
authors.forename: Lídia
authors.initials: L
authors.lastname: Barata
authors.forename: Marta
authors.initials: M
authors.lastname: Sousa Silva
authors.forename: Linda
authors.initials: L
authors.lastname: Schuldt
authors.forename: António E N
authors.initials: AE
authors.lastname: Ferreira
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Gomes
authors.forename: Ana M
authors.initials: AM
authors.lastname: Tomás
authors.forename: Manfred S
authors.initials: MS
authors.lastname: Weiss
authors.forename: Ana
authors.initials: A
authors.lastname: Ponces Freire
authors.forename: Carlos
authors.initials: C
authors.lastname: Cordeiro
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D018314:Leishmania infantum / Q000201:enzymology* / Q000235:genetics*; D013058:Mass Spectrometry; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000099301:Hydroxyacylglutathione Hydrolase
chemical_list: D005978:Glutathione; D013095:Spermidine; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase; C044809:trypanothione