The N - Terminal Cysteine - Rich Domain of Tobacco Class I Chitinase I s Essential for Chitin Binding but Not for Catalytic or Antifungal Activity ’ Beatrice Iseli

The vacuolar chitinases of class I possess an N-terminal cysteinerich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urfica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana fabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified to homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the Nterminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain i s a chitinbinding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for i ts substrate. The pH and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low pH values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD i s not necessary for catalytic or antifungal activity of chitinase.