Bipartite organization of the Bacillus subtilis endo-beta-1,4-glucanase revealed by C-terminal mutations.
The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-beta-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate. CD spectra indicated that the bulk of the alpha-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and approximately 4- to 5-fold higher with carboxymethylcellulose (soluble cellulose). These results along with data which show that EG470 binding capacity to microcrystalline cellulose is approximately 11 times more than that of EG300, demonstrate the importance of residues 330-499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330-499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine-326 and serine-321, respectively. Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.
Hefford
M A
MA
Institute of Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Laderoute
K
K
Willick
G E
GE
Yaguchi
M
M
Seligy
V L
VL
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D003505:Cyclodextrins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry
D003505:Cyclodextrins; D014312:Trisaccharides; C023872:mannotriose; D002480:Cellulase