Advertisement

Plant Molecular Biology

, Volume 71, Issue 3, pp 277–289 | Cite as

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

  • Wimal Ubhayasekera
  • Reetika Rawat
  • Sharon Wing Tak Ho
  • Malgorzata Wiweger
  • Sara Von Arnold
  • Mee-Len Chye
  • Sherry L. Mowbray
Article

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.

Keywords

Chitinase Family 19 Picea abies Norway spruce Conformational changes Class IV 

Notes

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).

References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Andersen OA, Dixon MJ, Eggleston IM, van Aalten DM (2005) Natural product family 18 chitinase inhibitors. Nat Prod Rep 22:563–579PubMedCrossRefGoogle Scholar
  3. Arlorio M, Ludwig A, Boller T, Bonfante P (1992) Inhibition of fungal growth by plant chitinases and beta-1, 3-glucanases—a morphological-study. Protoplasma 171:34–43CrossRefGoogle Scholar
  4. Berger S, Menudier A, Julien R, Karamanos Y (1995) Do de-N-glycosylation enzymes have an important role in plant cells? Biochimie 77:751–760PubMedCrossRefGoogle Scholar
  5. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242PubMedCrossRefGoogle Scholar
  6. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382:769–781PubMedCrossRefGoogle Scholar
  7. Brameld KA, Goddard WA 3rd (1998) The role of enzyme distortion in the single displacement mechanism of family 19 chitinases. Proc Natl Acad Sci USA 95:4276–4281PubMedCrossRefGoogle Scholar
  8. Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen rhizoctonia solani. Science 254:1194–1197PubMedCrossRefGoogle Scholar
  9. Brunner F, Stintzi A, Fritig B, Legrand M (1998) Substrate specificities of tobacco chitinases. Plant J 14:225–234PubMedCrossRefGoogle Scholar
  10. Collaborative Computational Project, Number 4 (1994) The CCP4 Suite: programs for protein crystallography. Acta Cryst D50:760–763Google Scholar
  11. Engh RA, Huber R (1991) Accurate bond and angle parameters for x-ray protein-structure refinement. Acta Crystallogr A 47:392–400CrossRefGoogle Scholar
  12. 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
  13. Frettinger P, Herrmann S, Lapeyrie F, Oelmuller R, Buscot F (2006) Differential expression of two class III chitinases in two types of roots of Quercus robur during pre-mycorrhizal interactions with Piloderma croceum. Mycorrhiza 16:219–223PubMedCrossRefGoogle Scholar
  14. Fukamizo T (2000) Chitinolytic enzymes: catalysis, substrate binding, and their application. Curr Protein Pept Sci 1:105–124PubMedCrossRefGoogle Scholar
  15. Hahn M, Hennig M, Schlesier B, Hohne W (2000) Structure of jack bean chitinase. Acta Crystallogr D Biol Crystallogr 56(Pt 9):1096–1099PubMedCrossRefGoogle Scholar
  16. Harris M, Jones TA (2001) Molray-a web interface between O and the POV-ray ray tracer. Acta Crystallogr D Biol Crystallogr 57:1201–1203PubMedCrossRefGoogle Scholar
  17. Hart PJ, Pfluger HD, Monzingo AF, Hollis T, Robertus JD (1995) The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 A resolution. J Mol Biol 248:402–413PubMedCrossRefGoogle Scholar
  18. Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino-acid-sequence similarities. Biochem J 293:781–788PubMedGoogle Scholar
  19. Hietala AM, Kvaalen H, Schmidt A, Johnk N, Solheim H, Fossdal CG (2004) Temporal and spatial profiles of chitinase expression by norway spruce in response to bark colonization by Heterobasidion annosum. Appl Environ Microbiol 70:3948–3953PubMedCrossRefGoogle Scholar
  20. Hoell IA, Dalhus B, Heggset EB, Aspmo SI, Eijsink VG (2006) Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes. FEBS J 273:4889–4900PubMedCrossRefGoogle Scholar
  21. Huet J, Rucktooa P, Clantin B, Azarkan M, Looze Y, Villeret V, Wintjens R (2008) X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases. Biochemistry 47:8283–8291PubMedCrossRefGoogle Scholar
  22. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47(Pt 2):110–119PubMedCrossRefGoogle Scholar
  23. Karasuda S, Tanaka S, Kajihara H, Yamamoto Y, Koga D (2003) Plant chitinase as a possible biocontrol agent for use instead of chemical fungicides. Biosci Biotechnol Biochem 67:221–224PubMedCrossRefGoogle Scholar
  24. Kasprzewska A (2003) Plant chitinases–regulation and function. Cell Mol Biol Lett 8:809–824PubMedGoogle Scholar
  25. Kawase T, Yokokawa S, Saito A, Fujii T, Nikaidou N, Miyashita K, Watanabe T (2006) Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2). Biosci Biotechnol Biochem 70:988–998PubMedCrossRefGoogle Scholar
  26. Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T, Nonaka T (2006) Structural studies of a two-domain chitinase from Streptomyces griseus HUT6037. J Mol Biol 358:472–484PubMedCrossRefGoogle Scholar
  27. Kim JS, Kim YO, Ryu HJ, Kwak YS, Lee JY, Kang H (2003) Isolation of stress-related genes of rubber particles and latex in fig tree (Ficus carica) and their expressions by abiotic stress or plant hormone treatments. Plant Cell Physiol 44:412–414PubMedCrossRefGoogle Scholar
  28. Kleywegt GJ, Jones TA (1996) Phi/psi-chology: Ramachandran revisited. Structure 4:1395–1400PubMedCrossRefGoogle Scholar
  29. 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
  30. 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
  31. Kraulis PJ (1991) Molscript—a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 24:946–950CrossRefGoogle Scholar
  32. 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
  33. Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33:491–497PubMedCrossRefGoogle Scholar
  34. McCarter JD, Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol 4:885–892PubMedCrossRefGoogle Scholar
  35. Meins F, Fritig B, Linthorst HJM, Mikkelsen JD, Neuhaus JM, Ryals J (1994) Plant chitinase genes. Plant Mol Biol Rep 12:S22–S28CrossRefGoogle Scholar
  36. Melchers LS, Apotheker-de Groot M, van der Knaap JA, Ponstein AS, Sela-Buurlage MB, Bol JF, Cornelissen BJ, van den Elzen PJ, Linthorst HJ (1994) A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. Plant J 5:469–480PubMedCrossRefGoogle Scholar
  37. Mizuno R, Fukamizo T, Sugiyama S, Nishizawa Y, Kezuka Y, Nonaka T, Suzuki K, Watanabe T (2008) Role of the loop structure of the catalytic domain in rice class I chitinase. J Biochem 143:487–495PubMedCrossRefGoogle Scholar
  38. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255PubMedCrossRefGoogle Scholar
  39. Neuhaus JM, Fritig B, Linthorst HJM, Meins F, Mikkelsen JD, Ryals J (1996) A revised nomenclature for chitinase genes. Plant Mol Biol Rep 14:102–104CrossRefGoogle Scholar
  40. 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
  41. Patil RS, Ghormade V, Deshpande MV (2000) Chitinolytic enzymes: an exploration. Enzyme Microb Technol 26:473–483PubMedCrossRefGoogle Scholar
  42. Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr 59:1131–1137PubMedCrossRefGoogle Scholar
  43. Robertus JD, Monzingo AF (1999) The structure and action of chitinases. EXS 87:125–135PubMedGoogle Scholar
  44. Schultze M, Staehelin C, Brunner F, Genetet I, Legrand M, Fritig B, Kondorosi E, Kondorosi A (1998) Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide nod signals. Plant J 16:571–580CrossRefGoogle Scholar
  45. Scorer CA, Clare JJ, McCombie WR, Romanos MA, Sreekrishna K (1994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Bio-Technology 12:181–184PubMedGoogle Scholar
  46. Song HK (1996) Refined structure of the chitinase from barley seeds at 2.0 A resolution. Acta Crystallogr D Biol Crystallogr 52:289–298PubMedCrossRefGoogle Scholar
  47. Tang CM, Chye ML, Ramalingam S, Ouyang SW, Zhao KJ, Ubhayasekera W, Mowbray SL (2004) Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1. Plant Mol Biol 56:285–298PubMedCrossRefGoogle Scholar
  48. Ubhayasekera W, Tang CM, Ho SW, Berglund G, Bergfors T, Chye ML, Mowbray SL (2007) Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops. FEBS J 274:3695–3703PubMedCrossRefGoogle Scholar
  49. Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Crystallogr 30:1022–1025CrossRefGoogle Scholar
  50. van Aalten DM, Komander D, Synstad B, Gaseidnes S, Peter MG, Eijsink VG (2001) Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci USA 98:8979–8984PubMedCrossRefGoogle Scholar
  51. van Hengel AJ, Tadesse Z, Immerzeel P, Schols H, van Kammen A, de Vries SC (2001) N-acetylglucosamine and glucosamine-containing arabinogalactan proteins control somatic embryogenesis. Plant Physiol 125:1880–1890PubMedCrossRefGoogle Scholar
  52. Vierheilig H, Alt-Hug M, Wiemken A, Boller T (2001) Hyphal in vitro growth of the arbuscular mycorrhizal fungus Glomus mosseae is affected by chitinase but not by beta-1,3-glucanase. Mycorrhiza 11:279–282CrossRefGoogle Scholar
  53. Wirth SJ, Wolf GA (1990) Dye-labelled substrates for the assay and detection of chitinase and lysozyme activity. J Microbiol Methods 12:197–205CrossRefGoogle Scholar
  54. 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
  55. Wright CS (1990) 2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose–wheat germ agglutinin isolectin complexes. J Mol Biol 215:635–651PubMedCrossRefGoogle Scholar
  56. Yan R, Hou J, Ding D, Guan W, Wang C, Wu Z, Li M (2008) In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi. J Basic Microbiol 48:293–301PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Wimal Ubhayasekera
    • 1
  • Reetika Rawat
    • 2
  • Sharon Wing Tak Ho
    • 2
  • Malgorzata Wiweger
    • 3
  • Sara Von Arnold
    • 4
  • Mee-Len Chye
    • 2
  • Sherry L. Mowbray
    • 1
  1. 1.Department of Molecular Biology, Biomedical CenterSwedish University of Agricultural SciencesUppsalaSweden
  2. 2.School of Biological SciencesThe University of Hong KongHong KongChina
  3. 3.Department of PathologyLeiden University Medical CenterLeidenThe Netherlands
  4. 4.Department of Plant Biology and Forest GeneticsSwedish University of Agricultural SciencesUppsalaSweden

Personalised recommendations