Chitinase B (ChiB) from Serratia marcescens is a family 18 exochitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrateassisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites ؊2 to ؉3) shows closure of the ''roof'' of the active site tunnel. It also shows that the sugar in the ؊1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the ؉1 and ؉2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.C hitinases hydrolyze chitin, a linear polymer of ␤-(1,4)-linked N-acetylglucosamine (NAG), which is an abundant biopolymer. These enzymes are essential to chitin-containing organisms (fungi, insects, crustaceans) and are used by several bacteria to exploit chitin as a source of energy. Chitinase inhibitors have generated a lot of interest given their potential as insecticides (1), fungicides (2, 3), and antimalarials (4, 5). Biotechnological exploitation of chitinases, as well as design of inhibitors with sufficiently high selectivity and affinity, requires detailed knowledge of the catalytic mechanism and enzyme-substrate interactions.Most nonplant chitinases belong to glycosidase family 18 (6) and degrade chitin with retention of the stereochemistry at the anomeric carbon (7-10). The reaction is thought to be initiated by distortion of the Ϫ1 sugar ring and protonation of the glycosidic oxygen by a protonated acidic residue. 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).Although the current model for the catalytic mechanism is well established, the amount of structural evidence in its support is limited (8). Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to f...

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Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (ba) 8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted )1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pK a calculations and available structural information. The results indicate that the pK a of the catalytic acid (Glu144) is ÔcycledÕ during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144.Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the )1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the )1 sugar and by increasing the pK a of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the )1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.

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