Substrate Distortion to a Boat Conformation at Subsite −1 Is Critical in the Mechanism of Family 18 Chitinases

Abstract

Using molecular dynamics simulations, we examined the plausible conformations for a hexaNAG substrate bound to the active site of Chitinase A. We find that (i) the hydrolysis mechanism of Chitinase A (a family 18 chitinase from Serratia marcescens) involves substrate distortion, (ii) the first step of acid-catalyzed hydrolysis (protonation of the linking anomeric oxygen between GlcNAc residues −1 and +1) requires a boat conformation for the GlcNAc residue at binding subsite −1; (iii) ab initio quantum mechanical calculations (HF/6-31G**) predict that protonation of a GlcNAc in a boat conformation leads to spontaneous anomeric bond cleavage to yield an oxazoline ion intermediate. We also studied several conformations of two possible hydrolysis intermediates: the oxocarbenium ion and the oxazoline ion. Only the oxazoline ion orients in the enzyme active site so as to allow stereoselective attack by water. This leads to retention of configuration in the anomeric product as observed experimentally. It is possible that all family 18 chitinases share a common mechanism. Hence, we suspect that distortion of the substrate into a boat form at subsite −1 is required for any glycosyl hydrolase which has only one acidic residue in the active site. The design of an inhibitor for these systems based on the boat distorted sugar conformation is discussed.

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

“…The sugar is in the boat conformation (Fig. 2), providing direct structural evidence for the distortion that was predicted on the basis of a family 20 glycosidase structure (9) and theoretical studies (15). Our EQ_NAG5 structure shows that the N-acetyl group is fixed in a conformation in which its oxygen is positioned only 3.0 Å from the anomeric carbon (C1) (Figs.…”

“…In both the WT (21) and the EQ structure an ordered water molecule is observed at hydrogen-bonding distance of the solvent-exposed Glu-144. Upon substrate binding, water molecules are displaced from the active site and Glu-144 is likely to be protonated (9,15). Rotation of the protonated Asp-142 stabilizes a deprotonated Glu-144 and thus promotes protonation of the glycosidic oxygen by the latter (Fig.…”

“…The subsequent nucleophilic attack differs from classical reaction mechanisms of retaining enzymes such as lysozyme (11) and amylases (12) in that it involves the N-acetyl group of the Ϫ1 sugar, rather than a carboxylate side chain on the protein (8,9,13,14). Thus, the first step of chitinolysis results in cleavage of the sugar chain and formation of an oxazolinium ion intermediate, and hydrolysis of this ion completes the reaction (9,15) (Fig. 1).…”

“…Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to family 18 (9,15,16). Furthermore, the model does not account for the roles of several highly conserved residues in family 18 chitinases that are known to be important for catalytic activity (17)(18)(19).…”

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

“…Previous studies suggest that family 18 enzymes initiate catalysis via distortion of the chitin substrate in the Ϫ1 subsite adjacent to the glycosidic bond (19,40,41). Substrate binding is accompanied by rotation of an aspartic acid (Asp-139 in ChiC; see below), which forms a hydrogen bond with the catalytic glutamic acid (Glu-141 in ChiC) and the N-acetyl group of the Ϫ1-bound sugar.…”