The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity.
The vacuolar chitinases of class I possess an N-terminal cysteine-rich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urtica 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 tabacum) 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 N-terminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitin-binding 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 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 is not necessary for catalytic or antifungal activity of chitinase.
Iseli
B
B
Botanisches Institut, Universität Basel, Switzerland.
Boller
T
T
Neuhaus
J M
JM
D000935:Antifungal Agents / Q000378:metabolism* / Q000633:toxicity; D001681:Biological Assay; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine; D017343:Genes, Plant; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008970:Molecular Weight; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014242:Trichoderma / Q000187:drug effects
D000935:Antifungal Agents; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002688:Chitinases; D003545:Cysteine