tailieunhanh - Báo cáo khoa học: Carbohydrate binding sites in Candida albicans exo-b-1,3-glucanase and the role of the Phe-Phe ‘clamp’ at the active site entrance

Candida albicansexo-b-1,3-glucanase (Exg; EC ) is implicated in cell wall b-d-glucan remodelling through its glucosyl hydrolase and⁄or transglucosylase activities. A pair of antiparallel phenylalanyl residues (F144 and F258) flank the entrance to the active site pocket. Various Exg mutants were studied using steady-state kinetics and crystallography aiming to understand the roles played by these residues in positioning the b-1,3-d-glucan substrate. | ỊFEBS Journal Carbohydrate binding sites in Candida albicans exo-p-1 3-glucanase and the role of the Phe-Phe clamp at the active site entrance Wayne M. Patrick1 2 Yoshio Nakatani1 z Susan M. Cutfield1 Miriam L. Sharpe1 Rochelle J. Ramsay3 and John F. Cutfield1 1 Department of Biochemistry University of Otago Dunedin New Zealand 2 Institute of NaturalSciences Massey University Auckland New Zealand 3 Institute of Molecular Biosciences Massey University Palmerston North New Zealand Keywords aromatic entranceway clamp exoglucanase glycoside hydrolase ordered water molecules protein-carbohydrate interaction site-directed mutagenesis Correspondence J. F. Cutfield Department of Biochemistry University of Otago PO Box 56 Dunedin 9054 New Zealand Fax 64 3 479 7866 Tel 64 3 479 7836 E-mail These authors contributed equally to this work Database Structural data for Exg mutants F258I F144Y F258Y and E292Q as wellas F229A E292S are available in the Protein Data Bank under the accession numbers 2PF0 3O6A 2PC8 and 3N9K respectively Received 17 August 2010 revised 1 September 2010 accepted 3 September 2010 doi Candida albicans exo-P-1 3-glucanase Exg EC is implicated in cell wall P-D-glucan remodelling through its glucosyl hydrolase and or transglucosylase activities. A pair of antiparallel phenylalanyl residues F144 and F258 flank the entrance to the active site pocket. Various Exg mutants were studied using steady-state kinetics and crystallography aiming to understand the roles played by these residues in positioning the P-1 3-D-glucan substrate. Mutations at the Phe-Phe entranceway demonstrated the requirement for double-sided CH p interactions at the 1 subsite and the necessity for phenylalanine rather than tyrosine or tryptophan. The Tyr-Tyr double mutations introduced ordered water molecules into the entranceway. A third Phe residue F229 nearby was evaluated as a possible 2 subsite. The inactive double .