An elaboration on the syn–anti proton donor concept of glycoside hydrolases: electrostatic stabilisation of the transition state as a general strategy

Abstract

An in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the À1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrateÕs ring-oxygen at the transition state. This is substantially different depending on whether the enzymeÕs proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed.

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

“…Such a transition state would place the O2 hydroxyl group of the relevant mannoside pseudo-equatorial and avoid troublesome steric clashes at the anomeric centre. In contrast, some authors have advocated a 3 H 4 transition state for retaining ␤-mannosidase catalysis on theoretical grounds (36). Such a transition state, however, would require O4 to become pseudo axial and thus demand a major, unprecedented conformational change in the enzyme in order to prevent steric clashes of the Ϫ2 subsite sugar.…”

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello‐oligomers show high flexibility in the substrate binding

“…One alternative is, of course, that one route is dominant and other routes are side routes. There were no signs of higher energy conformations (Nerinckx et al 2005;Stam et al 2005) of the glycosyl ring at subsite À1 in the determined complex structures, but, of course, this does not exclude the possibility that some glucosyl units of the substrate would bind to the active site tunnel in a distorted conformation.…”

Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds

“…21,51,73 And proximality of the C2 hydroxyl group towards the nucleophile is also obtained; such interaction may be important in different β-glucoside hydrolases. 16,76 The apparent strict xylan preference by GH11 enzymes may then not be achieved through an exotic reaction itinerary but through a simple steric incompatibility towards cellulose.…”

“…13 Current understanding of glycosidase mechanisms has been reviewed thoroughly, [14][15][16][17] and recently three new general concepts have been put forward: 1) the syn/anti proton donor positioning; 12 2) the presence of a hydrophobic platform as a common feature within the -1 subsite; 18 and 3) enzymes with syn-positioned proton donors perform electrostatic transition state (TS) stabilization (known to be the most important factor for enzymatic catalysis 19,20 ) towards the substrate's ring oxygen by means of the conjugate base of their proton donor, whereas antiprotonating enzymes provide a separate electron-rich residue for this purpose. 21 The considerations in this work pertain to pyranoside glycosidic bond substitutions that operate by a classical exocyclic mechanism on oxyalkyl-type leaving groups, thus not to: 1) the endocyclic mechanism 22 which has not yet been observed with glycoside hydrolases; 2) the SN isubstitution mechanism that occurs in glycosyl fluoride solvolysis 23 and in-situ anomerisations with good leaving groups, 24 and has been proposed to be operative in retaining glycosyltransferases; 25 3) catalysis by GH family 4 (GH4) glycosidases that operate by a redox and elimination mechanism; 26 and 4) GH families 18, 20 (Clan K) 27,28 and 84 29 that utilize anchimeric assistance from the substrates' N-acetyl group, resulting in a strained oxazoline-type glycosyl intermediate (chitinases/chitobiases). Based on its orientation relative to a reference atom in the Fisher projection (IUPAC nomenclature), an α/β glycosidic bond often -but not always -corresponds with an axial/equatorial substituent orientation in the ground state conformation.…”

“…This can only influence K ass and be of relatively minor importance for TS stabilization, which is mainly achieved through electrostatic compensation by the enzyme of the change in charges 19,20 occurring at the O5 ring oxygen. 21 Thus, if a B 2,5 -TS belongs to the substitution itinerary within the tested β-glycoside hydrolases (from families 1, 3, 6, 7 and 18), one should expect the locked-boat disaccharide 5 to be a substrate or a relatively good inhibitor -which is never the case. Boat conformations have nevertheless been proposed as transition states within the mechanism of several glycoside hydrolases on the basis of observed conformers within the liganded complexes, most notably within GH26 50 and 11.…”