TAILIEUCHUNG - Báo cáo khoa học: Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops

Brassica junceachitinase is an endo-acting, pathogenesis-related protein that is classified into glycoside hydrolase family 19, with highest homology (50–60%) in its catalytic domain to class I plant chitinases. Here we report X-ray structures of the chitinase catalytic domain from wild-type (apo,as well as with chloride ions bound) and a Glu234Ala mutant enzyme, solved by molecular replacement and refined at , and A˚ resolution, respectively. | ỊFEBS Journal Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops Wimal Ubhayasekera1 Ce Mun Tang2 Sharon W. T. Ho2 Gunnar Berglund3 t Terese Bergfors3 Mee-Len Chye2 and Sherry L. Mowbray1 1 Department of Molecular Biology Swedish University of AgriculturalSciences Uppsala Sweden 2 Department of Botany The University of Hong Kong Pokfulam Hong Kong 3 Department of Celland Molecular Biology Uppsala University Sweden Keywords Brassica juncea chitinase conformational changes endochitinase family 19 Correspondence S. L. Mowbray Department of Molecular Biology Box 590 BiomedicalCenter Swedish University of AgriculturalSciences SE-751 24 Uppsala Sweden Fax 46 18 53 6971 Tel 46 18 471 4990 E-mail mowbray@ Website http Present address Department of Biology Hong Kong University of Science and Technology Clearwater Bay Kowloon Hong Kong tSkeisvang Videregaende Skole Haugesund Norway Brassica juncea chitinase is an endo-acting pathogenesis-related protein that is classified into glycoside hydrolase family 19 with highest homology 50-60 in its catalytic domain to class I plant chitinases. Here we report X-ray structures of the chitinase catalytic domain from wild-type apo as well as with chloride ions bound and a Glu234Ala mutant enzyme solved by molecular replacement and refined at and A resolution respectively. Confirming our earlier mutagenesis studies the active-site residues are identified as Glu212 and Glu234. Glu212 is believed to be the catalytic acid in the reaction whereas Glu234 is thought to have a dual role both activating a water molecule in its attack on the anomeric carbon and stabilizing the charged intermediate. The molecules in the various structures differ significantly in the conformation of a number of loops that border the active-site cleft. The differences suggest an opening and closing of the enzyme during the catalytic cycle. Chitin is expected to dock first

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