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



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García Oliva, Cecilia María and Hoyos Vidal, Pilar and Petrásková, Lucie and Kulik, Natalia and Pelantová, Helena and Cabanillas, Alfredo H. and Rumbero, Ángel and Křen, Vladimír and Hernáiz Gómez-Dégano, María José and Bojarová, Pavla (2019) Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation. International Journal of Molecular Sciences, 20 (24). p. 6181. ISSN 1422-0067

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Fungal 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.

Item Type:Article
Uncontrolled Keywords:β-N-acetylhexosaminidases; substrate specificity; transglycosylation; Glide docking; Talaromyces flavus; muramic acid; non-reducing carbohydrate
Subjects:Medical sciences > Pharmacy > Pharmaceutical chemistry
ID Code:67123
Deposited On:20 Jul 2021 14:48
Last Modified:17 Aug 2021 08:46

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