Peroxyl radicals modify 6-phosphogluconolactonase from Escherichia coli via oxidation of specific amino acids and aggregation which inhibits enzyme activity

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dc.catalogadorjwg
dc.contributor.authorReyes Valenzuela, Juan Sebastián
dc.contributor.authorFuentes Lemus, Eduardo Felipe
dc.contributor.authorRomero, Jefferson
dc.contributor.authorArenas, Felipe
dc.contributor.authorFierro Huerta, Angelica María
dc.contributor.authorDavies, Michael J.
dc.contributor.authorLopez Alarcon Camilo Ignacio
dc.date.accessioned2025-01-02T18:04:06Z
dc.date.available2025-01-02T18:04:06Z
dc.date.issued2023
dc.description.abstract6-phosphogluconolactonase (6PGL) catalyzes the second reaction of the pentose phosphate pathway (PPP) converting 6-phosphogluconolactone to 6-phosphogluconate. The PPP is critical to the generation of NADPH and metabolic intermediates, but some of its components are susceptible to oxidative inactivation. Previous studies have characterized damage to the first (glucose-6-phosphate dehydrogenase) and third (6-phosphogluconate dehydrogenase) enzymes of the pathway, but no data are available for 6PGL. This knowledge gap is addressed here. Oxidation of Escherichia coli 6PGL by peroxyl radicals (ROO center dot, from AAPH (2,2 '-azobis(2-methylpropionamidine) dihydrochloride) was examined using SDS-PAGE, amino acid consumption, liquid chromatography with mass detection (LC-MS), protein carbonyl formation and computational methods. NADPH generation was assessed using mixtures all three enzymes of the oxidative phase of the PPP. Incubation of 6PGL with 10 or 100 mM AAPH resulted in protein aggregation mostly due to reducible (disulfide) bonds. High fluxes of ROO center dot induced consumption of Cys, Met and Trp, with the Cys oxidation rationalizing the aggregate formation. Low levels of carbonyls were detected, while LC-MS analyses provided evidence for oxidation of selected Trp and Met residues (Met1, Trp18, Met41, Trp203, Met220 and Met221). ROO center dot elicited little loss of enzymatic activity of monomeric 6PGL, but the aggregates showed diminished NADPH generation. This is consistent with in silico analyses that indicate that the modified Trp and Met are far from the 6-phosphogluconolactone binding site and the catalytic dyad (His130 and Arg179). Together these data indicate that monomeric 6PGL is a robust enzyme towards oxidative inactivation by ROO center dot and when compared to other PPP enzymes.
dc.fuente.origenORCID
dc.identifier.doi10.1016/j.freeradbiomed.2023.04.019
dc.identifier.eissn1873-4596
dc.identifier.issn0891-5849
dc.identifier.urihttps://doi.org/10.1016/j.freeradbiomed.2023.04.019
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/89460
dc.identifier.wosidWOS:001001503000001
dc.information.autorucEscuela de Química; Lopez Alarcon Camilo Ignacio; S/I; 1004308
dc.information.autorucEscuela de Química; Fierro Huerta Angelica Maria; 0000-0002-6507-4188; 218137
dc.information.autorucEscuela de Química; Fuentes Lemus Eduardo Felipe; 0000-0002-1465-8466; 186720
dc.information.autorucEscuela de Química; Reyes Valenzuela Juan Sebastian; S/I; 1268701
dc.language.isoen
dc.nota.accesocontenido completo
dc.pagina.final127
dc.pagina.inicio118
dc.publisherELSEVIER SCIENCE INC
dc.revistaFree Radical Biology and Medicine
dc.rightsacceso restringido
dc.subject6-phosphogluconolactonase
dc.subjectPentose phosphate pathway
dc.subjectAAPH
dc.subjectPeroxyl radicals
dc.subjectProtein oxidation
dc.subjectAggregation
dc.subjectMethionine oxidation
dc.subjectTryptophan oxidation
dc.subjectCysteine oxidation
dc.subject.ddc570
dc.subject.deweyBiologíaes_ES
dc.titlePeroxyl radicals modify 6-phosphogluconolactonase from Escherichia coli via oxidation of specific amino acids and aggregation which inhibits enzyme activity
dc.typeartículo
dc.volumen204
sipa.codpersvinculados1004308
sipa.codpersvinculados218137
sipa.codpersvinculados186720
sipa.codpersvinculados1268701
sipa.trazabilidadORCID;2024-12-23
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