Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase

Item Type:	Article
Title:	Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase
Creators Name:	Hahn, M. and Olsen, O. and Politz, O. and Borriss, R. and Heinemann, U.
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.
Keywords:	Amino Acid Sequence, Aspartic Acid, Bacillus, Base Sequence, Binding Sites, Escherichia Coli, Glutamic Acid, Glycoside Hydrolases, Hydrogen Bonding, Molecular Cloning, Molecular Models, Molecular Sequence Data, Oligodeoxyribonucleotides, Point Mutation, Protein Conformation, Recombinant Proteins, Secondary Protein Structure, Site-Directed Mutagenesis, Tryptophan, X-Ray Crystallography
Source:	Journal of Biological Chemistry
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Volume:	270
Number:	7
Page Range:	3081-3088
Date:	17 February 1995
Official Publication:	https://doi.org/10.1074/jbc.270.7.3081
PubMed:	View item in PubMed

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