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The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

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Abstract

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.

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Acknowledgments

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).

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Authors and Affiliations

  1. Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, P.O. Box 590, 751 24, Uppsala, Sweden

    Wimal Ubhayasekera & Sherry L. Mowbray

  2. School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China

    Reetika Rawat, Sharon Wing Tak Ho & Mee-Len Chye

  3. Department of Pathology, Leiden University Medical Center, L1-Q, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden, The Netherlands

    Malgorzata Wiweger

  4. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, P.O. Box 7080, 75007, Uppsala, Sweden

    Sara Von Arnold

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  1. Wimal Ubhayasekera
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  2. Reetika Rawat
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  4. Malgorzata Wiweger
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Correspondence to Wimal Ubhayasekera.

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Ubhayasekera, W., Rawat, R., Ho, S.W.T. et al. The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce. Plant Mol Biol 71, 277–289 (2009). https://doi.org/10.1007/s11103-009-9523-9

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