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

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-Angstrom resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp(101) Glu(103), Asp(105), and Glu(107) 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 Glu(103) and Glu(107) 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 Glu(103) and Glu(107) are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule.