Abstract
The conformational itineraries taken by carbohydrate residues in the catalytic subsite of retaining glycoside hydrolases (GHs), harness the link between substrate conformation and reactivity. GHs’ active sites may be described as a combination of subsites dedicated to the binding of individual sugar residues and to catalysis. The three-dimensional structure of GH:carbohydrate complexes has demonstrated that carbohydrate ring conformation changes in an ordered manner during catalysis. Here we demonstrate in silico that a link exists between subsite binding dynamics and substrate specificity for β-galactosidases from clan GH-A families GH1, GH2, GH35, GH42 and GH59. Different oligosaccharides were docked in the active site of reference β-galactosidase structures using Vina-Carb. Subsequent molecular dynamics (MD) simulations revealed that these enzymes favor a high degree of flexibility and ring distortion of the substrate the lytic subsite −1. Although the β-galactosidase families examined are structurally and mechanistically related, distinct patterns of ring distortion were unveiled for the different families. For β-galactosidases, three different family-dependent reaction itineraries (1S3 → 4H3‡ → 4C1, 1,4B → 4H3/ 4E‡ → 4C1, and 1S5 → 4E/ 4H5‡ → 4C1) were identified, all compatible with the antiperiplanar lone pair hypothesis (ALPH) for the hydrolysis of β-glycosides. This comparative study reveals the fuzzy character of the changes in carbohydrate ring geometry prior to carbohydrate hydrolysis.
Highlights
Β-Galactosidases (E.C. 3.2.1.23) catalyze the hydrolysis of terminal non-reducing end β-D-galactosyl residues found in oligosaccharides and glycoconjugates
Some families like GH1 and GH5 have shown remarkable plasticity in their ability to act on a large variety of residues, one third of the families are relatively specific as they act only on a single type of substrates. β-Linked D-glucosyl, D-galactosyl, D-mannosyl and D-xylosyl residues constitute the major types of cleaved residues of clan glycoside hydrolases (GHs)-A enzymes
The conserved protein structure and catalytic machinery within clan GH-A have evident plastic properties that allowed to generate an extraordinary diversity of enzymes able to cleave equatorially-linked glycans either in an exo- or in an endo-manner
Summary
Β-Galactosidases (E.C. 3.2.1.23) catalyze the hydrolysis of terminal non-reducing end β-D-galactosyl residues found in oligosaccharides and glycoconjugates. Β-Galactosidases can be obtained directly from microbial sources and from plants and animals This enzyme activity is heavily applied industrially to reduce the lactose content in dairy products, the transglycosylating properties of some enzymes being employed for the production of galactooligosaccharides (GOS)[1,2,3]. The structure of several protein-carbohydrate complexes revealed a range of non-ground state ring geometries within subsite −1 of the active site[9,10]. These have been used to describe intermediate conformational forms along the reaction pathway and have led to the definition of key steps in the reaction pathway of retaining β-glycosidases[11,12,13]. The conformational particularities of the cleaved residues affect the resulting reaction paths
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