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

0519717 - MBÚ 2020 RIV CH eng J - Článek v odborném periodiku
Garcia-Oliva, C. - Hoyos, P. - Petrásková, Lucie - Kulik, Natalia - Pelantová, Helena - Cabanillas, A.H. - Rumbero, A. - Křen, Vladimír - Hernáiz, M.J. - Bojarová, Pavla
Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation.
International Journal of Molecular Sciences. Roč. 20, č. 24 (2019), č. článku 6181. E-ISSN 1422-0067
Grant CEP: GA MŠMT(CZ) LTC19038; GA ČR(CZ) GA18-01163S
Výzkumná infrastruktura: CESNET II - 90042; CERIT-SC - 90085
Institucionální podpora: RVO:61388971
Klíčová slova: Glide docking * Talaromyces flavus * muramic acid
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 4.556, rok: 2019
Způsob publikování: Open access
https://www.mdpi.com/1422-0067/20/24/6181

Fungal Beta-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The Beta-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 Beta-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-Beta-d-glucosaminide or 4-nitrophenyl N-acetyl-Beta-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 Beta(1-4) position, the galacto-acceptor afforded a Beta(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on Beta-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.
Trvalý link: http://hdl.handle.net/11104/0304716