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Biochemical, genetic and primary sequence analyses of the Erwinia chrysanthemi
endoglucanase EGZ allowed us to identify two functional domains and to locate
their boundaries. The catalytic domain extends from residue 1 to 288, while a
domain required for EGZ to bind to microcrystalline cellulose lies from residues
324 to 385. Each domain was found capable of functioning in the absence of the
other. A region rich in Pro, Thr, and Ser residues links both domains and
appeared to be susceptible to proteolytic attack. Based upon predictions derived
from a method developed to compare sequences sharing a low level of similarity,
e.g. hydrophobic cluster analysis (HCA), we analysed the importance of either
residue His98 or Glu133 in EGZ catalytic activity. Two EGZ-derived proteins were
engineered in which either His98 or Glu133 amino acid was converted to an Ala
residue. Characterization of the purified proteins showed that no enzymatic
activity could be detected, by using carboxymethylcellulose (CMC) or
paranitrophenyl-cellobioside (pNPC) as substrates, while both mutated proteins
retained the capacity to bind to microcrystalline cellulose. These studies,
which to date constitute the first experimental testing of HCA-derived
predictions, allowed us to identify two particular amino acids involved in
cellulolytic activity. By taking into account data from chemical modification
studies of other cellulases, we speculate that the His98 residue is involved in
the folding of the catalytic domain while the Glu133 residue intervenes directly
in the beta, 1-4 glycosidic bond cleavage.
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