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Volume 156, Issue 12

Crystal structure and mutagenesis analysis of chitinase CrChi1 from the nematophagous fungus in complex with the inhibitor caffeine Free

Abstract

Chitinases are a group of enzymes capable of hydrolysing the -(1,4)-glycosidic bonds of chitin, an essential component of the fungal cell wall, the shells of nematode eggs, and arthropod exoskeletons. Chitinases from pathogenic fungi have been shown to be putative virulence factors, and can play important roles in infecting hosts. However, very limited information is available on the structure of chitinases from nematophagous fungi. Here, we present the 1.8 Å resolution of the first structure of a Family 18 chitinase from this group of fungi, that of CrChi1, and the 1.6 Å resolution of CrChi1 in complex with a potent inhibitor, caffeine. Like other Family 18 chitinases, CrChi1 has the DXDXE motif at the end of strand 5, with Glu174 as the catalytic residue in the middle of the open end of the (/) barrel. Two caffeine molecules were shown to bind to CrChi1 in subsites −1 to +1 in the substrate-binding domain. Moreover, site-directed mutagenesis of the amino acid residues forming hydrogen bonds with caffeine molecules suggests that these residues are important for substrate binding and the hydrolytic process. Our results provide a foundation for elucidating the catalytic mechanism of chitinases from nematophagous fungi and for improving the pathogenicity of nematophagous fungi against agricultural pest hosts.

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Keyword(s): CAZY, carbohydrate-active enzymes database , PDB, Protein Data Bank and rms, root mean square
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2010-12-01
2024-04-19
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References

  1. Åhman J., Johanson T., Olsson M., Punt P. J., van den Hondel C. A. M. J. J., Tunlid A. S. 2002; Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Appl Environ Microbiol 68:3408–3415
     [Google Scholar]
  2. Arai N., Shiomi K., Yamaguchi Y., Masuma R., Iwai Y., Turberg A., Koelbl H., Omura S. 2000; Argadin, a new chitinase inhibitor, produced by Clonostachys sp. FO-7314. . Chem Pharm Bull (Tokyo) 48:1442–1446
     [Google Scholar]
  3. Bortone K., Monzingo A. F., Ernst S., Robertus J. D. 2002; The structure of an allosamidin complex with the Coccidiodes immitis defines a role for second acid residue in substrate assisted mechanism. J Mol Biol 320:293–302
     [Google Scholar]
  4. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M. other authors 1998; Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921
     [Google Scholar]
  5. DeLano W. 2002 The PyMol Molecular Graphics System San Carlos, CA, USA: DeLano Scientific;
     [Google Scholar]
  6. Emsley P., Cowtan K. 2004; Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132
     [Google Scholar]
  7. Esnouf R. M. 1997; An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J Mol Graph Model 15:132–134
     [Google Scholar]
  8. Fusetti F., von Moeller H., Houston D., Rozeboom H. J., Dijkstra B. W., Boot R. G., Aerts J. M., van Aalten D. M. F. 2002; Structure of human chitotriosidase. Implications for specific inhibitor design and function of mammalian chitinase-like lectins. J Biol Chem 277:25537–25544
     [Google Scholar]
  9. Gan Z. W., Yang J. K., Tao N., Liang L. M., Mi Q. L., Li J., Zhang K. Q. 2007a; Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita . Appl Microbiol Biotechnol 76:1309–1317
     [Google Scholar]
  10. Gan Z. W., Yang J. K., Tao N., Yu Z. F., Zhang K. Q. 2007b; Cloning and expression analysis of a chitinase gene Crchi1 from the mycoparasitic fungus Clonostachys rosea (syn. Gliocladium roseum ). J Microbiol 45:422–430
     [Google Scholar]
  11. Gan Z. W., Yang J. K., Tao N., Lou Z. Y., Mi Q. L., Meng Z. H., Zhang K. Q. 2009; Crystallization and preliminary crystallographic analysis of a chitinase from Clonostachys rosea . Acta Crystallogr Sect F Struct Biol Cryst Commun 65:386–388
     [Google Scholar]
  12. Gooday G. W., Zhu W. Y., O'Donnell R. W. 1992; What are the roles of chitinases in the growing fungus. FEMS Microbiol Lett 100:387–391
     [Google Scholar]
  13. Hollis T., Monzingo A. F., Bortone K., Ernst S., Cox R., Robertus J. D. 2000; The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis . Protein Sci 9:544–551
     [Google Scholar]
  14. Houston D. R., Eggleston I., Synstad B., Eijsink V. G., van Aalten D. M. F. 2002; The cyclic dipeptide C1-4[cyclo-(l-Arg-d-Pro)] inhibits family 18 chitinases by structural mimicry of a reaction intermediate. Biochem J 368:23–27
     [Google Scholar]
  15. Hurtado-Guerrero R., van Aalten D. M. F. 2007; Structure of Saccharomyces cerevisiae chitinase 1 and screening-based discovery of potent inhibitors. Chem Biol 14:589–599
     [Google Scholar]
  16. Kuranda M. J., Robbins P. W. 1991; Chitinase is required for cell separation during growth of Saccharomyces cerevisiae . J Biol Chem 266:19758–19767
     [Google Scholar]
  17. Laskowski R., MacArthur M., Moss D., Thornton J. 1993; procheck: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291
     [Google Scholar]
  18. Li J., Yang J. K., Huang X. W., Zhang K. Q. 2006; Purification and characterization of an extracellular protease from Clonostachys rosea and its potential as a pathogenic factor. Process Biochem 41:925–929
     [Google Scholar]
  19. Lorito M., Mach R. L., Sposato P., Strauss J., Peterbauer C. K., Kubicek C. P. 1996; Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum . Proc Natl Acad Sci U S A 93:14868–14872
     [Google Scholar]
  20. Mamarabadi M., Jensen B., Lübeck M. 2008; Three endochitinase-encoding genes identified in the biocontrol fungus Clonostachys rosea are differentially expressed. Curr Genet 54:57–70
     [Google Scholar]
  21. Matthews B. W. 1968; Solvent content of protein crystals. J Mol Biol 33:491–497
     [Google Scholar]
  22. McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., Winn M. D., Storoni L. C., Read R. J. 2007; Phaser crystallographic software. J Appl Crystallogr 40:658–674
     [Google Scholar]
  23. Meng Z. H., Lou Z. Y., Liu Z., Hui D., Bartlam M., Rao Z. H. 2006; Purification, characterization, and crystallization of human pyrroline-5-carboxylate reductase. Protein Expr Purif 49:83–87
     [Google Scholar]
  24. Merritt E. A., Murphy M. E. 1994; Raster3D version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr 50:869–873
     [Google Scholar]
  25. Omura S., Arai N., Yamaguchi Y., Masuma R., Iwai Y., Namikoshi M., Turberg A., Kölbl H., Shiomi K. 2000; Argifin, a new chitinase inhibitor, produced by Gliocladium sp. FTD-0668.. J Antibiot (Tokyo) 53:603–608
     [Google Scholar]
  26. Otwinowski Z., Minor W. 1997; Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326
     [Google Scholar]
  27. Papanikolau Y., Tavlas G., Vorgias C. E., Petratos K. 2003; De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin. Acta Crystallogr D Biol Crystallogr 59:400–403
     [Google Scholar]
  28. Rajamohan F., Alzate O., Cotrill J. A., Curtiss A., Dean D. H. 1996; Protein engineering of Bacillus thuringiensis δ -endotoxin: mutations at domain II of Cry1Ab enhance receptor affinity and toxicity towards gypsy moth larvae. Proc Natl Acad Sci U S A 93:14338–14343
     [Google Scholar]
  29. Rao F. V., Andersen O. A., Vora K. A., DeMartino J. A., van Aalten D. M. F. 2005a; Methylxanthine drugs are chitinase inhibitors: investigation of inhibition and binding modes. Chem Biol 12:973–980
     [Google Scholar]
  30. Rao F. V., Houston D. R., Boot R. G., Aerts J. M., Hodkinson M., Adams D. J., Shiomi K., Omura S., van Aalten D. M. F. 2005b; Specificity and affinity of natural product cyclopentapeptide inhibitors against A. fumigatus , human, and bacterial chitinases. Chem Biol 12:65–76
     [Google Scholar]
  31. Schüttelkopf A. W., Andersen O. A., Rao F. V., Allwood M., Lloyd C., Eggleston I. M., van Aalten D. M. F. 2006; Screening-based discovery and structural dissection of a novel family 18 chitinase inhibitor. J Biol Chem 281:27278–27285
     [Google Scholar]
  32. Seidl V., Huemer B., Seiboth B., Kubicek C. P. 2005; A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases. FEBS J 272:5923–5939
     [Google Scholar]
  33. Sutton J. C., Li D. W., Peng G., Yu H., Zhang P., Valdebenito-Sanhueza R. M. 1997; Gliocladium roseum a versatile adversary of a Botrytis cinerea in crops. Plant Dis 81:316–328
     [Google Scholar]
  34. Terwisscha van Scheltinga A. C., Armand S., Kalk K. H., Isogai A., Henrissat B., Dijkstra B. W. 1995; Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substract assisted catalysis. Biochemistry 34:15619–15623
     [Google Scholar]
  35. van Aalten D. M. F., Komander D., Synstad B., Gàseidnes S., Peter M. G., Eijsink V. G. H. 2001; Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci U S A 98:8979–8984
     [Google Scholar]
  36. Xue A. G. 2003; Biological control of pathogens causing root rot complex in field pea using Clonostachys rosea strain ACM941. Phytopathology 93:329–335
     [Google Scholar]
  37. Yang J. K., Tian B. Y., Liang L. M., Zhang K. Q. 2007; Extracellular enzymes and the pathogenesis of nematophagous fungi. Appl Microbiol Biotechnol 75:21–31
     [Google Scholar]
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Crystal structure and mutagenesis analysis of chitinase CrChi1 from the nematophagous fungus Clonostachys rosea in complex with the inhibitor caffeine
Microbiology 156, 3566 (2010); https://doi.org/10.1099/mic.0.043653-0
/content/journal/micro/10.1099/mic.0.043653-0
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