Bipartite organization of the Bacillus subtilis endo-{beta}-1,4-glucanase revealed by C-terminal mutations |
| |
Authors: | Hefford, Mary Alice Laderoute, Keith Willick, Gordon E. Yaguchi, Makoto Seligy, Verner L. |
| |
Affiliation: | 1institute of Biological Sciences, National Research Council of Canada 2Animal Research Centre Agriculture Canada Ottawa, Ontario, Canada K1A 0R6 |
| |
Abstract: | The C-terminal boundary of primary sequence of the Bacillussubtilis PAP115 endo-ß-1,4-glucanase (EG) requiredfor stable catalytic activity has been mapped by site-directedmutagenesis using Escherichia coli as host. The 52 kDa cel geneproduct, EG470 and a 33 kDa mutant (EG300), lacking 170 residuesthrough a nonsense mutation at the leucine-330 codon of thegene, exhibited similar patterns of enzymatic activity and pHoptima using cellooligopentaose as substrate.CD spectra indicatedthat the bulk of the -helical secondary structure in EG470 wascontained within EG300. However, relative to EG470, the specificactivity of EG300 was 3- to 4-fold lower with amorphous celluloseas substrate and {small tilde}4-to5-fold higher with carboxymethylcellulose(soluble cellulose).These results along with data which showthat EG470 binding capacity to mirocrystalline cellulose is{small tilde} 11 times more than that of EG300, demonstratethe importance of residues 330499 for non-catalytic bindingof cellulose. A construct of the cel gene carrying a deletionof codons 330499 and an insertion of a nonsense codonat leucine-330, was further used to make mutants EG296 and EG291with nonsense codon substitutions at arginine and serine-321,respectively.Western analysis using EG-specific antiserum revealedthat relative losses in enzymatic activity of EG296 (50%) andEG291 (95%) could be accounted for by the extent of their proteolysis,signifying a marked destabilization of these enzymes by removalof only a few amino acids. |
| |
Keywords: | Bacillus subtilis/ catalytic and cellulose binding domains/ circular dichroism/ deletion mutagenesis/ endoglucanase sequence |
本文献已被 Oxford 等数据库收录! |
|