RT Journal Article
T1 Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
A1 García Oliva, Cecilia María
A1 Hoyos Vidal, María Pilar
A1 Petrásková, Lucie
A1 Kulik, Natalia
A1 Pelantová, Helena
A1 Cabanillas, Alfredo H.
A1 Rumbero, Ángel
A1 Křen, Vladimír
A1 Hernáiz Gómez-Degano, María Josefa
A1 Bojarová, Pavla
AB 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.
PB MDPI
SN 1422-0067
YR 2019
FD 2019-12-07
LK https://hdl.handle.net/20.500.14352/8320
UL https://hdl.handle.net/20.500.14352/8320
LA eng
NO Ministerio de Economía, Comercio y Empresa (España)
NO MInisterio de Educación, Juventud y Deporte (República Checa)
NO Czech Science Foundation
DS Docta Complutense
RD 4 abr 2025