The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce
Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.
KeywordsChitinase Family 19 Picea abies Norway spruce Conformational changes Class IV
The authors would like to thank Dr. Fred Asiegbu (Swedish University of Agricultural Sciences) for providing Heterobasidion annosum (strain FP5), and Dr. Mark Harris (Uppsala University) for photographing the chitinase-inhibited fungal plate. The work was supported by the Swedish Research Council (VR) and the Swedish Foundation for Strategic Research via the Glycoconjugates in Biological Systems network, GLIBS (SLM), as well as by the University of Hong Kong (ORA10208034) (MLC).
- Collaborative Computational Project, Number 4 (1994) The CCP4 Suite: programs for protein crystallography. Acta Cryst D50:760–763Google Scholar
- Evans PR (1997) Scaling of MAD data. In: Wilson KS, Davies G, Ashton AW, Bailey S (eds) Proceedings of the CCP4 study weekend. CCLRC Daresbury Laboratory, Warrington, pp 97–102Google Scholar
- Kleywegt GJ, Jones TA (1997) Detecting folding motifs and similarities in protein structures. In: Carter JCW, Sweet RM (eds) Methods in enzymology, macromolecular crystallography, Pt B. Academic Press, New York, pp 525–545Google Scholar
- Kleywegt GJ, Zou JY, Kjeldgaard M, Jones TA (2001) Around O. In: Rossmann MG, Arnold E (eds) International tables for crystallography. Vol. F crystallography of biological macromolecules. Kluwer, Dordrecht, pp 353–356, 366–367Google Scholar
- Leslie AGW (1992) Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB newsletter on protein crystallographyGoogle Scholar
- Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. In: Carter JCW, Sweet RM (eds) Methods in enzymology, macromolecular crystallography, Pt A. Academic Press, New York, pp 307–326Google Scholar
- Wiweger M, Farbos I, Ingouff M, Lagercrantz U, Von Arnold S (2003) Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. J Exp Bot 54:2691–2699PubMedCrossRefGoogle Scholar