Substrate specificity and antifungal activity of recombinant tobacco class I chitinases.
Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.
Suarez
V
V
Friedrich Miescher Institute, Novartis Research Foundation, Basel, Switzerland.
Staehelin
C
C
Arango
R
R
Holtorf
H
H
Hofsteenge
J
J
Meins
F
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010457:Peptidoglycan / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000187:drug effects / Q000254:growth & development
D000935:Antifungal Agents; D008070:Lipopolysaccharides; C092082:Nod factor IV, Rhizobium meliloti; D010457:Peptidoglycan; D010940:Plant Proteins; D011994:Recombinant Proteins; C023023:lipid-linked oligosaccharides; D002686:Chitin; D002688:Chitinases