Structural basis for transglycosylation in glycoside hydrolase family GH116 glycosynthases
| Autor: | S. Pengthaisong, Yanling Hua, James R. Ketudat Cairns |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular 0301 basic medicine Cellobiose Anomer Glycoside Hydrolases Protein Conformation Stereochemistry Biophysics Oligosaccharides Crystallography X-Ray Biochemistry Substrate Specificity Ligases Nitrophenols 03 medical and health sciences Hydrolysis chemistry.chemical_compound Catalytic Domain Glycoside hydrolase Glycosides Molecular Biology Ternary complex chemistry.chemical_classification 030102 biochemistry & molecular biology biology Glycoside Active site Glycosynthase Glucose 030104 developmental biology Amino Acid Substitution chemistry Mutation biology.protein Thermodynamics Thermoanaerobacterium Protein Binding |
| Zdroj: | Archives of Biochemistry and Biophysics. 706:108924 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/j.abb.2021.108924 |
| Popis: | Glycosynthases are glycoside hydrolase mutants that can synthesize oligosaccharides or glycosides from an inverted donor without hydrolysis of the products. Although glycosynthases have been characterized from a variety of glycoside hydrolase (GH) families, family GH116 glycosynthases have yet to be reported. We produced the Thermoanaerobacterium xylanolyticum TxGH116 nucleophile mutants E441D, E441G, E441Q and E441S and compared their glycosynthase activities to the previously generated E441A mutant. The TxGH116 E441G and E441S mutants exhibited highest glycosynthase activity to transfer glucose from α-fluoroglucoside (α-GlcF) to cellobiose acceptor, while E441D had low but significant activity as well. The E441G, E441S and E441A variants showed broad specificity for α-glycosyl fluoride donors and p-nitrophenyl glycoside acceptors. The structure of the TxGH116 E441A mutant with α-GlcF provided the donor substrate complex, while soaking of the TxGH116 E441G mutant with α-GlcF resulted in cellooligosaccharides extending from the +1 subsite out of the active site, with glycerol in the −1 subsite. Soaking of E441A or E441G with cellobiose or cellotriose gave similar acceptor substrate complexes with the nonreducing glucosyl residue in the +1 subsite. Combining structures with the ligands from the TxGH116 E441A with α-GlcF crystals with that of E441A or E441G with cellobiose provides a plausible structure of the catalytic ternary complex, which places the nonreducing glucosyl residue O4 2.5 A from the anomeric carbon of α-GlcF, thereby explaining its apparent preference for production of β-1,4-linked oligosaccharides. This functional and structural characterization provides the background for development of GH116 glycosynthases for synthesis of oligosaccharides and glycosides of interest. |
| Databáze: | OpenAIRE |
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