Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

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dc.contributor.authorGarcía Oliva, Cecilia María
dc.contributor.authorHoyos Vidal, Pilar
dc.contributor.authorPetrásková, Lucie
dc.contributor.authorKulik, Natalia
dc.contributor.authorPelantová, Helena
dc.contributor.authorCabanillas, Alfredo H.
dc.contributor.authorRumbero, Ángel
dc.contributor.authorKřen, Vladimír
dc.contributor.authorHernáiz Gómez-Dégano, María José
dc.contributor.authorBojarová, Pavla
dc.date.accessioned2023-06-17T09:10:16Z
dc.date.available2023-06-17T09:10:16Z
dc.date.issued2019-12-07
dc.description.abstractFungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.
dc.description.departmentDepto. de Química en Ciencias Farmacéuticas
dc.description.facultyFac. de Farmacia
dc.description.refereedTRUE
dc.description.sponsorshipMinisterio de Economía y Competitividad (MINECO)
dc.description.sponsorshipMinistry of Education, Youth and Sports of the Czech Republic
dc.description.sponsorshipCzech Science Foundation
dc.description.statuspub
dc.eprint.idhttps://eprints.ucm.es/id/eprint/67123
dc.identifier.doi10.3390/ijms20246181
dc.identifier.issn1422-0067
dc.identifier.officialurlhttps://doi.org/10.3390/ijms20246181
dc.identifier.relatedurlhttps://www.mdpi.com/1422-0067/20/24/6181
dc.identifier.urihttps://hdl.handle.net/20.500.14352/8320
dc.issue.number24
dc.journal.titleInternational Journal of Molecular Sciences
dc.language.isoeng
dc.page.initial6181
dc.publisherMDPI
dc.relation.projectIDRTI2018-096037-B-I00
dc.relation.projectIDLTC19038
dc.relation.projectID18-01163S
dc.rightsAtribución 3.0 España
dc.rights.accessRightsopen access
dc.rights.urihttps://creativecommons.org/licenses/by/3.0/es/
dc.subject.keywordβ-N-acetylhexosaminidases
dc.subject.keywordsubstrate specificity
dc.subject.keywordtransglycosylation
dc.subject.keywordGlide docking
dc.subject.keywordTalaromyces flavus
dc.subject.keywordmuramic acid
dc.subject.keywordnon-reducing carbohydrate
dc.subject.ucmQuímica farmaceútica
dc.subject.unesco2390 Química Farmacéutica
dc.titleAcceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
dc.typejournal article
dc.volume.number20
dspace.entity.typePublication
relation.isAuthorOfPublicationd6ff8ba2-55f0-40b4-ac38-4edb93669e9d
relation.isAuthorOfPublication.latestForDiscoveryd6ff8ba2-55f0-40b4-ac38-4edb93669e9d
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