Abstract
The reaction mechanism of glycoside hydrolases belonging to family 1 (GH1) of carbohydrate-active enzymes classification, hydrolysing β-O-glycosidic bonds, is well characterised. This family includes several thousands of enzymes with more than 20 different EC numbers depending on the sugar glycone recognised as substrate. Most GH1 β-glycosidases bind their substrates with similar specificity through invariant amino acid residues. Despite extensive studies, the clear identification of the roles played by each of these residues in the recognition of different glycones is not always possible. We demonstrated here that a histidine residue, completely conserved in the active site of the enzymes of this family, interacts with the C2-OH of the substrate in addition to the C3-OH as previously shown by 3 D-structure determination.
Highlights
glycoside hydrolases belonging to family 1 (GH1) is one of the largest families of the carbohydrate active enzymes classification data bank CAZy, including more than 26,000 b-glycosidases, of which more than 300 have been characterised with the resolution of more than 60 3 D-structures solved and over 20 different substrates specificities identified
GH1 enzymes follow the classical retaining reaction mechanism proposed by Koshland[17]
We evaluated the function of this amino acid by combining site-directed mutagenesis, the use of mechanism-based inhibitors and thermodynamic cycles on the well studied b-glycosidase from Sulfolobus solfataricus (Ssb-gly)
Summary
GH1 is one of the largest families of the carbohydrate active enzymes classification data bank CAZy (http://www.cazy. org1), including more than 26,000 b-glycosidases, of which more than 300 have been characterised with the resolution of more than 60 3 D-structures solved and over 20 different substrates specificities identified. The catalytic mechanisms and substrate binding sites of GH1 b-glycosidases have been the subject of extensive studies through enzymological characterisation performed with mechanism-based inhibitors and site-directed mutants, the inspection of 3 D-structures in the presence of substrate analogues and inhibitors, computational analysis, and quantum mechanics[20,21,22,23,24,25] These approaches have helped define the roles of the invariant residues in the active sites of GH1 as either catalytic residues or in substrate binding. We evaluated the function of this amino acid by combining site-directed mutagenesis, the use of mechanism-based inhibitors and thermodynamic cycles on the well studied b-glycosidase from Sulfolobus solfataricus (Ssb-gly) For this enzyme, which shows broad substrate specificity toward gluco-, galacto, and fucosides, high resolution 3 D-structures with different inhibitors and detailed enzymatic studies are already available[2,29,30,31,32]. Of the nucleophile of the reaction (E387) and interacts with C2OH, which could not be determined via the inspection of the 3 D-structure
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