Crystal structures of the cellulase Ce148F in complex with inhibitors and substrates give insights into its processive action

Cellulase Ce148F from Clostridium cellulolyticum was described as a process ive endo-cellulase. The active site is composed of a 25 Angstrom long tunne l which is followed by an open cleft. During the processive action, the cel lulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage. To st udy this processive action in the tunnel, the native catalytic module of Ce 148F and the inactive mutant E55Q, have been cocrystallized with cellobiito l, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oli gosaccharides cellobiose, -tetraose and -hexaose, Seven sub-sites in the tu nnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed, The sub-sites observed for the thio-oligosaccharide inhibitors and oligosac charides, respectively, were located at two different positions in the tunn el corresponding to a shift in the chain direction of about a half sugar su bunit. These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel. Multiple patterns are not ob served in nonprocessive endo-cellulases, where only one sugar position is f avored by aromatic stacking. It is therefore proposed that the aromatic res idues serve as lubricating agents to reduce the sliding barrier in the proc essive action.