SafchiA is a New Class of Defence Chitinase from Saffron (Crocus sativus L.)

Plant chitinases (EC 3.2.1.14) catalyse the hydrolysis of chitin, a β-1, 4-linked homopolymer of GlcNac. Chitinases are widely distributed in the plant kingdom and have diverse roles in plant growth and development, as well as in defence responses. We have isolated a chitinase from saffron (Crocus sativus L.) named as SafchiA. A lot of chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. The deduced SafchiA protein share high similarities with chitinases belong to family 19 of glycosyl-hydrolases. Although, some changes in the enzyme catalytic domain are present. The most relevant difference is that one glutamic acid in the enzyme active site described as essential in catalytic event of family 19 glycosyl-hydrolases is not present in SafchiA. Instead a glutamic acid SafchiA posses a tyrosine residue at the corresponding position (Tyr136). In order to gain detailed insight in the role of this and other residues in C. sativus chitinase activity, we have developed a heterologous expression system for SafchiA and different mutated versions of its catalytic domain in E. coli. Using barley class II chitinase as a paradigm, site directed mutagenesis of different active site residues was performed. The abilities of wild type recombinant SafchiA catalytic domain and different mutated versions to hydrolyse the soluble high-molecular-mass substrate CM-chitin-RBV were investigated. Results showed that it seems like only one glutamic acid residue is necessary for the enzyme activity instead of two described in family 19 chitinases.

Keywords:

defence , new chitinase , site-directed mutagenesis