Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds.
Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.
Departamento de Biotecnologia, E.T.S. Ingenieros de Montes, Universidad Politecnica de Madrid, E-28040 Madrid, Spain.
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Garcia-Casado
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Collada
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Allona
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Casado
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Pacios
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Aragoncillo
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Gomez
D000595:Amino Acid Sequence; D000935:Antifungal Agents; D001483:Base Sequence; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014197:Trees / Q000201:enzymology*
D000935:Antifungal Agents; D017931:DNA Primers; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases