1MAC

CRYSTAL STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF BACILLUS MACERANS ENDO-1,3-1,4-BETA-GLUCANASE

Summary for 1MAC

• Entry DOI: 10.2210/pdb1mac/pdb
• Descriptor: 1,3-1,4-BETA-D-GLUCAN 4-GLUCANOHYDROLASE, CALCIUM ION (3 entities in total)
• Functional Keywords: hydrolase (glucanase)
• Biological source: Paenibacillus macerans
• Total number of polymer chains: 2
• Total formula weight: 47732.62

Authors

Hahn, M.,Heinemann, U. (deposition date: 1994-12-22, release date: 1995-02-27, Last modification date: 2024-11-13)

Primary citation

Hahn, M.,Olsen, O.,Politz, O.,Borriss, R.,Heinemann, U.
Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase.
J.Biol.Chem., 270:3081-3088, 1995

PubMed Abstract: In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases). Here, the relationship between structure and activity of the beta-glucanase of Bacillus macerans is studied by x-ray crystallography and site-directed mutagenesis of active site residues. Crystal structure analysis at 2.3-A resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp101, Glu103, Asp105, and Glu107 as in the hybrid Bacillus beta-glucanase H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5287-5291). Different mutant proteins with substitutions in these residues are generated by site-directed mutagenesis, isolated, and characterized. Compared with the wild-type enzyme their activity is reduced to less than 1%. Several mutants with isosteric substitutions in Glu103 and Glu107 are completely inactive, suggesting a direct role of these residues in glycosyl bond hydrolysis. The kinetic properties of mutant beta-glucanases and the crystal structure of the wild-type enzyme are consistent with a mechanism where Glu103 and Glu107 are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule.

PubMed: 7852389
DOI: 10.1074/jbc.270.7.3081

Experimental method

X-RAY DIFFRACTION (2.3 Å)