Intrinsic dynamic behavior of enzyme:substrate complexes govern the catalytic action of β-galactosidases across clan GH-A

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 ( 1 S 3 → 4 H 3 ‡ → 4 C 1 , 1,4 B → 4 H 3 / 4 E ‡ → 4 C 1 , and 1 S 5 → 4 E/ 4 H 5 ‡ → 4 C 1 ) 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.

Recent advances in β-galactosidase and fructosyltransferase immobilization technology

“…Calculations also suggest that a change on the galactosyl ring to a halfchair conformation is also important for the glycosidic bond cleavage (Br as, Fernandes, and Ramos 2010). This ring conformation is consistent on all GH-A enzymes, although differences between families have been found (Kumar, Henrissat, and Coutinho 2019).…”

“…Crystallographic and kinetic studies confirm this finding (Zechel and Withers 2000). All enzymes from the GH-A super family are structurally and mechanistically related (Kumar, Henrissat, and Coutinho 2019).…”

Xyloglucan Oligosaccharides Hydrolysis by Exo-Acting Glycoside Hydrolases from Hyperthermophilic Microorganism Saccharolobus solfataricus

“…Therefore, XylS requires the removal of glucose and galactose residues at the non-reducing end to access to the following xyloside residues ( Figure 1 ). LacS, like other GH1 enzymes [ 34 ], has broad substrate specificity, promoting the hydrolysis of 1,4- and 1,2-β- d -glycosides [ 23 , 35 , 36 ]. Moreover, an in silico docking study by Kumar and co-workers suggested that a LacS active site could well accommodate short-branched XGOs (GLG) with β-galactose at non-reducing termini in the subsite -1, and the interaction with XGOs could be stronger and selective compared to that of the XG-active β-galactosidase of C. japonicus [ 35 ].…”

Homodimerization of a glycoside hydrolase family GH1 β‐glucosidase suggests distinct activity of enzyme different states

“…Identification of the molecular features responsible for the increase in catalytic efficiency upon Sfβgly homodimerization could deepen the comprehension of the oligomerization and activity relationship. For instance, if enzyme dynamics dictate the performance of substrate cleavage, 43 then differences in the dynamic ensemble between monomeric and dimeric states would be expected. Naturally, study of the physiological importance of oligomers in native organism ( S. frugiperda ) could shed light on the biology of these insects.…”