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

• Published in: International journal of molecular sciences 2019-12, Vol.20 (24), p.6181
• Main Authors: Garcia-Oliva, Cecilia, Hoyos, Pilar, Petrásková, Lucie, Kulik, Natalia, Pelantová, Helena, Cabanillas, Alfredo H, Rumbero, Ángel, Křen, Vladimír, Hernáiz, María J, Bojarová, Pavla
• Format: Article
• Language: English
• Subjects: Acids; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - metabolism; Chromatography; Data acquisition; Disaccharides; Enzymes; Galactose; Glucose; Glycosides; Glycosides - metabolism; Glycosylation; Histidine; Kinetics; Models, Molecular; Mutation; N-Acetylgalactosamine; N-Acetylglucosamine; Oligosaccharides; Oligosaccharides - metabolism; Protein Conformation; Spectrum analysis; Structure-Activity Relationship; Substrate Specificity; Talaromyces; Talaromyces - enzymology; Uronic acid
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms20246181

Fungal β- -acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β- -acetylhexosaminidase from is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in 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- -acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl -acetyl-β-d-glucosaminide or 4-nitrophenyl -acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features ( -acetylglucosamine, glucose, -acetylgalactosamine, galactose, -acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 -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 -configuration at C-4 steered the glycosylation into the β(1-4) position, the -acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and -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 -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.