par Collet, Laetitia;Denys, Agathe ;Oudjama, Yamina;Sifennasr, Kawtar;Wauven, Corinne Vander;Dutoit, Raphaël
Référence Acta Crystallographica Section D: Structural Biology, 82, page (12)
Publication Publié, 2026-05-11
Article révisé par les pairs
Résumé : Glycoside hydrolases (GHs) achieve glycan breakdown through glycosidic bondhydrolysis. Some retaining GHs can also transglycosylate, which could be usefulfor glycosynthesis. Improving GHs for either glycan degradation or synthesisrequires a deep understanding of the residues involved in catalysis and substratebinding. This study characterizes in detail the activity and structure ofTa_Cel5A, a cellulase in glycoside hydrolase family 5, subfamily 5 (GH5_5) fromthe thermophilic ascomycete Thermoascus aurantiacus. While its hydrolyticactivity was confirmed, Ta_Cel5A was also found to exhibit a weak transglycosylase activity with cellopentaose as a substrate. Transglycosylation productswere detected within the first minutes of reaction at 25�C, far below its optimaltemperature. The structures of catalytically impaired variants were solved incomplex with oligosaccharides. The entire catalytic cleft was defined, consistingof seven glucose-binding subsites, five negative subsites and two positivesubsites, from the nonreducing end to the reducing end. The fifth negativesubsite could not be inferred in silico, showing the limitation in predicting distalsubsites based on structural analogy. The structure of the glycosyl-enzymeintermediate was also obtained, revealing the displacement of key residues inthe active site. The covalent binding of a glycosidic molecule triggers a majordisplacement of the nucleophilic residue, Glu244, changing its interactionnetwork. The acid/base residue, Glu133, and a conserved tyrosine residue,Tyr201, are also displaced during glycosyl-enzyme intermediate formation,hinting at their role in the deglycosylation step.

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