Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis.
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.
Laboratoire de Chimie Bactérienne, CNRS, Marseille, France.
B
B
Py
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Bortoli-German
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J
Haiech
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M
Chippaux
F
F
Barras
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002872:Chromosome Deletion; D004885:Erwinia / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment
D005971:Glutamates; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D002480:Cellulase