THE REFINED CRYSTAL-STRUCTURE OF AN ENDOCHITINASE FROM HORDEUM-VULGARE L SEEDS AT 1.8 ANGSTROM RESOLUTION

Class II chitinases (EC 3.2.1.14) are plant defense proteins. They hyd rolyze chitin, an insoluble beta-1,4-linked polymer of N-acetylglucosa mine (NAG), which is a major cell-wall component of many fungal hyphae . We previously reported the three-dimensional structure of the 26 kDa class II endochitinase from barley seeds at 2.8 Angstrom resolution, determined using multiple isomorphous replacement (MIR) methods. Here, we report the crystallographic refinement of this chitinase structure against data to 1.8 Angstrom resolution using rounds of hand rebuildi ng coupled with molecular dynamics (X-PLOR). The final model has an X- value of 18.1% for the 5.0 to 1.8 Angstrom data shell and 19.8% for th e 10.0 to 1.8 Angstrom shell, and root-mean-square deviations from sta ndard bond lengths and angles of 0.017 Angstrom and 2.88 degrees, resp ectively The 243 residue molecule has one beta-sheet, ten alpha-helice s and three disulfide bonds; 129 water molecules are included in the f inal model. We show structural comparisons confirming that chitinase s econdary structure resembles lysozyme at the active site region. Based on substrate binding to lysozyme, we have built a hypothetical model for the binding of a hexasaccharide into the pronounced active site cl eft of chitinase. This provides the first view of likely substrate int eractions from this family of enzymes; the model is consistent with a lysozyme-like mechanism of action in which Glu67 acts as proton donor and Glu89 is likely to stabilize the transition state oxycarbonium ion . These binding site residues, and many hydrophobic residues are conse rved in a range of plant chitinases. This endochitinase structure will serve as a model for other plant chitinases, and that catalytic model s based on this structure will be applicable to the entire enzyme fami ly.