Surface residue mutations which change the substrate specificity of Thermomonospora fusca endoglucanase E2.

The three dimensional structure of a T. fusca endoglucanase catalytic domain (E2cd) has been determined by X-ray crystallography at 1.0 A resolution (Wilson et al., 1995). The availability of a high resolution structure for E2cd allows us to initiate structure-based efforts to engineer cellulases with a high activity on native cellulose. The low activity on crystalline cellulose suggests that the entry of a cellulose molecule into the active site rather than catalysis may be the rate limiting step for hydrolysis of crystalline cellulose. Movement of a loop upon substrate binding has been proposed to play a crucial role in catalysis. A total of 15 surface mutants and 5 loop mutants were created by site-directed mutagenesis and their effects on activity and substrate specificity were determined. Circular dichroism spectra were used to monitor structural changes, and no major changes were found. The binding constants for two methyl umbelliferyl oligosaccharides and cellotriose were measured for some of the mutants and all of them showed binding similar to wild type E2. These results provide the first direct link between loop movement and catalysis by E2 and show that surface residues can affect its substrate specificity.