STRUCTURAL CHARACTERIZATION OF CHITINASE

Abstract

A protein (CHT) with chitinase activity was purified from the seeds of Tamarindus indica (Tamarind) by three step column chromatography involving an affinity and two ion exchange matrices. CHT was found to be an abundant protein of tamarind seed kernel. The molecular mass of the protein was determined to be 34 kDa by SDS-PAGE and SEC analysis. The protein was also showed to be a glycoprotein. The protein could be concentrated to 15 mg/ml. It is the characteristic of class III chitinases to have an antifungal and lysozyme activity but CHT lacked both antifungal and lysozyme activity. The enzyme was shown to be activated in presence of EDTA and monovalent ions. The activity was inhibited in the presence of divalent cations and reducing agents. N-terminal sequencing showed significant homology to class III chitinase (class III chitinase from V. vinifera). The partial CHT cDNA and gene was amplified using PCR. The gene was demonstrated to have introns. The PCR product from genomic DNAand mRNAgave a product of size ~ 730bp in length. The product was sequenced. Comparison of the retrieved sequence showed 61% identity to class III chitinase from Glycine max (from nr database) and 57% identity to hevamine from Hevea brasielinsis ( from pdb repository). Fluorescence study for CHT was done using denaturing conditions of temperature, PME, urea and guanidium chloride. In presence of urea and GndCl, there was considerable denaturation of the CHT although formation of hydrophobic pockets was not observed when urea and GndCl was used in presence of ANS. HCl tend to denature the protein completely above 1.2 MHCl. Higher temperature tend to cause a VI red shift in the tryptophan fluorescence intensity curve indicating in total unfolding of the protein and exposure of buried tryptophan residues. CHT was crystallized in the presence of PEG4000 at pH 6. The crystal belonged to tetragonal P4i space group and diffracted to 2.6 AD. The unit-cell parameters a = b = 67, c = 173.09 A. The crystals contain two molecules in asymmetric unit which corresponds to a crystal volume per unit molecular weight (VM) of 2.50 AD3 Da"1. The structure was solved by molecular replacement method using the structure of hevamine from H. brasienlinsis as a search model. After series ofrefinement cycles, the model was fitted well in electron density and was refined to R factor of 28.6% with overall correlation coefficient of 84.13%. Structural analysis showed an overall conservation of the topology belonging to class III chitinases. CHT has an apg barrel structure with a substrate binding groove between the two chains. Each chain has three disulphide bridges and hydrogen bonding network which stabilizes the conserved residues present in the active site of the protein. Docking studies using HEX indicated that Tetra-NAG was fitting well in the substrate binding groove of CHT with an E-value of-273.71 vn