The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity.
We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.
Baird
S D
SD
Division of Biological Sciences, National Research Council of Canada.
Hefford
M A
MA
Johnson
D A
DA
Sung
W L
WL
Yaguchi
M
M
Seligy
V L
VL
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005971:Glutamates; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D005971:Glutamates; D002480:Cellulase