Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi.
The catalytic domains of beta-1,4-glucanases can be grouped into families of related amino acid sequences. The endoglucanase CenA from Cellulomonas fimi is a member of family B. All enzymes from this family are believed to hydrolyze beta-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond. Three-dimensional structures for two cellulases from family B have been determined by X-ray crystallographic analysis. These structures show that there are four Asp residues which are in a position to function as acid catalyst, base catalyst, and/or transition state stabilizers. These aspartates are conserved in all members of family B. The roles of Asp216, Asp252, Asp287, and Asp392, the corresponding amino acids in CenA, were determined. These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis, and the resulting effect on activity, substrate specificity, and overall structure has been determined. Changes in overall structure were monitored using circular dichroism spectroscopy, and no significant differences between the wild-type and mutant proteins were found. Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column. The Michaelis-Menten parameters of the enzyme were determined on 2,4-dinitrophenyl cellobioside as well as (carboxymethyl)-cellulose and phosphoric acid-swollen cellulose. Initial characterization of mutant proteins indicates that Asp252 and Asp392 are the acid and base catalysts, respectively, in CenA. Residue Asp287 appears to aid Asp252 in acid catalysis, and Asp216 is not absolutely required for catalysis.
Department of Chemistry,University of British Columbia, Vancouver, Canada.
H G
HG
Damude
S G
SG
Withers
D G
DG
Kilburn
R C
RC
Miller
R A
RA
Warren
D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D017931:DNA Primers; D001224:Aspartic Acid; D002480:Cellulase