Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.
Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
W W
WW
Wakarchuk
R L
RL
Campbell
W L
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Sung
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J
Davoodi
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M
Yaguchi
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase
D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase