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

Chitinase B (Chill) from Serratia marcescens is a family 18 exo-chitinase w hose catalytic domain has a TIM-barrel fold with a tunnel-shaped active sit e. We have solved structures of three Chill complexes that reveal details o f substrate binding, substrate-assisted catalysis, and product displacement . The structure of an inactive Chill mutant (E144Q) complexed with a pentam eric substrate (binding in subsites -2 to +3) shows closure of the "roof" o f the active site tunnel. It also shows that the sugar in the -1 position i s distorted to a boat conformation, thus providing structural evidence in s upport 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 sho w 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 cha nges in the core of the TIM barrel that involve conserved polar residues wh ose functions were hitherto unknown. These changes simultaneously contribut e to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.