Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis

Biochemical, genetic and primary sequence analyses of the Erwiniachrysanthemi endoglucanase EGZ allowed us to identify two functionaldomains and to locate their boundaries. The catalytic domainextends from residue 1 to 288, while a domain required for EGZto bind to microcrystalline cellulose lies from residues 324to 385. Each domain was found capable of functioning in theabsence of the other. A region rich in Pro, Thr and Ser residueslinks both domains and appeared to be susceptible to proteolyticattack. Based upon predictions derived from a method developedto compare sequences sharing a low level of similarity, e.g.hydrophobic cluster analysis (HCA), we analysed the importanceof either residue His98 or Glul33 in EGZ catalytic activity.Two EGZ-derived proteins were engineered in which either His98or Glul33 amino acid was converted to an Ala residue. Characterizationof the purified proteins showed that no enzymatic activity couldbe detected, by using carboxymethylcellulose (CMC) or paranitrophenyl-cellobioside(pNPC) as substrates, while both mutated proteins retained thecapacity to bind to microcrystalline cellulose. These studies,which to date constitute the first experimental testing of HCA-derivedpredictions, allowed us to identify two particular amino acidsinvolved in cellulolytic activity. By taking into account datafrom chemical modification studies of other cellulases, we speculatethat the His98 residue is involved in the folding of the catalyticdomain while the Glul33 residue intervenes directly in the ß,1–4 glycosidic bond cleavage.