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 t o 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 is 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 its substrate. The p H 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 p H 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.

Bacterial, fungalG animal, and some plant ehNnases form fmily 18 of glycosyl hydrelases. Most plant ehitinases form the family 19, While sense ¢kitinases also have lysozyme activity, animal ~mzymes belong to different families. For 81ycesyl hydrobmes, two reaction mechanisms are possible, lendin8 to either retention or inversion of the anomerlc conflauratlon. We analyzed by HPLC the stereoehemleal ontcome of the hydrolysis catalyzed by cucumber and bean chitlnaes~ belonging to families 18 and 19, respectively. Cucumber ehltlnue used the reteJnlng mechanism as known for bacterial cldtlmmeJ and hen eag white lysozyme for which the mechanlmn hun been determined. In contrast, bean ¢ltltinase utalyzed the hydrolysis of cldtoollgosaeehafldes with overall inversion of anomerle configuration.Key words: Chitinase; Lysozyme; Catalytic mechanism; Anomeri¢ configuration I, Introduction Chitinases (EC 3.2.1.14) are enzymes that catalyze the hydrolysis of 13-1,4 linkages in chitin, a homopolymer of N-acetyl-~glucosamine [1]. The substrates of chitinases and lysozymes are very similar. Indeed, some chitinases also hydrolyse bacterial cell walls [1,2].Plant chitinases are a structurally diverse group with respect to their physical properties, enzymatic activities and localization [3]. Some chitinases, especially in combination with [3-1,3 glucanases, inhibit fungal growth in vitro [4], substantiating the idea that these enzymes play an important role in plant defense against pathogens, Chitinases could also release elicitors from funlpd cell walls [5], Recently it was shown that root chitinases can differentially inactivate Nod-factors produced by certain P, ht=obtmn strains and therefore may determine the specificity of tl,e bacterium-host plant interaction as well as modulate the activity of Nod-factors [6]. Chitinase may also have an important function in development, as demonstrated by the ability of a chitinase to overcome a block in embryogenesis in a mutant cell line of carrot [7]. Plant chitinases have been divided into several classes based on amino acid sequence [8], In the classification system of glycosyl hydrolases, all chitinases are grouped into two families [9]. Class I, II, IV and V chitinases form the family 19, that is only known in plants. Family 18 includes all fungal, animal and bacterial chitinases as well as plant chitinases of classes III and VI.The hydrolytic action of glycosyl hydrolases can take place with either retention or inversion of the anomeric configuration. The hydrolytic mechanism of but a few chitinases has been analyzed. Two bacterial chitinases and hevamme, a plant chitinase/lysozyme displayed a retaining mechanism [10,11]. Hen egg white lysozyme (family 22) [12] is also a retaining enzyme, while T4 lysozyme (family 24) was recently found to invert the anomeric configuration [13]. Holm and Sander [14] recently proposed that a family 19 chitinase and several lysozymes descend from a common ancestor, based on a weak folding similarity and the similar function. Goose lysozyme (Ya...

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