Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide Nod signals

The ability of 16 chitinases from seven different plant species to hydrolyz e a collection of several structurally related lipochitooligosaccharides (L COs) of Rhizobium was analyzed. It was found that the enzymes differed to a large extent in their activity on different LCOs. Differences were attribu ted to (i) the relative activity on different LCOs as substrate (e.g, sulfa ted versus non-sulfated LCOs); (ii) the relative cleavage site preference o n a given LCO molecule (hydrolysis of either the second, third or fourth gl ycosidic bond from the non-reducing end of the molecule); and (iii) the ste reochemistry of the reaction (retention or inversion of the anomeric config uration). A graphic representation of the different substrate specificities resulted in a 'fingerprint' that is characteristic for a given enzyme or a family of related enzymes. By comparing the LCO-fingerprint of unknown enz ymes with those obtained for already characterized proteins, it is possible to identify new glycosyl hydrolases. The high diversity of substrate speci ficity found among plant chitinases may reflect variations in the natural s ubstrates of the enzymes, such as substitutions on the chitin moiety of fun gal cell walls or, in plants, the presence of putative endogenous substrate s related to LCOs.