Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-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-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.
Forschungsgruppe Kristallographie, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Federal Republic of Germany.
M
M
Hahn
O
O
Olsen
O
O
Politz
R
R
Borriss
U
U
Heinemann
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli; D018698:Glutamic Acid; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014364:Tryptophan
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D014364:Tryptophan; D006026:Glycoside Hydrolases; C028020:licheninase