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. 1994 Jul;176(13):3885–3894. doi: 10.1128/jb.176.13.3885-3894.1994

Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85.

H Zhu 1, F W Paradis 1, P J Krell 1, J P Phillips 1, C W Forsberg 1
PMCID: PMC205585  PMID: 8021170

Abstract

The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen J. D., Thoma J. A. Model for carbohydrase action. Aspergillus oryzae alpha-amylase degradation of maltotriose. Biochemistry. 1978 Jun 13;17(12):2345–2350. doi: 10.1021/bi00605a014. [DOI] [PubMed] [Google Scholar]
  2. Allen J. D., Thoma J. A. Multimolecular substrate reactions catalyzed by caabohydrases. Aspergillus oryzae alpha-amylase degradation of maltooligosaccharides. Biochemistry. 1978 Jun 13;17(12):2338–2344. doi: 10.1021/bi00605a013. [DOI] [PubMed] [Google Scholar]
  3. Antoniou T., Marquardt R. R., Cansfield P. E. Isolation, partial characterization, and antinutritional activity of a factor (pentosans) in rye grain. J Agric Food Chem. 1981 Nov-Dec;29(6):1240–1247. doi: 10.1021/jf00108a035. [DOI] [PubMed] [Google Scholar]
  4. Biely P., Krátký Z., Vrsanská M. Substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus. Eur J Biochem. 1981 Oct;119(3):559–564. doi: 10.1111/j.1432-1033.1981.tb05644.x. [DOI] [PubMed] [Google Scholar]
  5. Biely P., Vrsanská M., Krátký Z. Mechanisms of substrate digestion by endo-1,4-beta-xylanase of Cryptococcus albidus. Lysozyme-type pattern of action. Eur J Biochem. 1981 Oct;119(3):565–571. doi: 10.1111/j.1432-1033.1981.tb05645.x. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Bray M. R., Clarke A. J. Action pattern of xylo-oligosaccharide hydrolysis by Schizophyllum commune xylanase A. Eur J Biochem. 1992 Feb 15;204(1):191–196. doi: 10.1111/j.1432-1033.1992.tb16623.x. [DOI] [PubMed] [Google Scholar]
  8. Bray M. R., Clarke A. J. Essential carboxy groups in xylanase A. Biochem J. 1990 Aug 15;270(1):91–96. doi: 10.1042/bj2700091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clarke A. J. Essential tryptophan residues in the function of cellulase from Schizophyllum commune. Biochim Biophys Acta. 1987 Apr 30;912(3):424–431. doi: 10.1016/0167-4838(87)90048-3. [DOI] [PubMed] [Google Scholar]
  10. Deshpande V., Hinge J., Rao M. Chemical modification of xylanases: evidence for essential tryptophan and cysteine residues at the active site. Biochim Biophys Acta. 1990 Nov 15;1041(2):172–177. doi: 10.1016/0167-4838(90)90062-k. [DOI] [PubMed] [Google Scholar]
  11. Forsberg C. W., Beveridge T. J., Hellstrom A. Cellulase and Xylanase Release from Bacteroides succinogenes and Its Importance in the Rumen Environment. Appl Environ Microbiol. 1981 Nov;42(5):886–896. doi: 10.1128/aem.42.5.886-896.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gebler J., Gilkes N. R., Claeyssens M., Wilson D. B., Béguin P., Wakarchuk W. W., Kilburn D. G., Miller R. C., Jr, Warren R. A., Withers S. G. Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases. J Biol Chem. 1992 Jun 25;267(18):12559–12561. [PubMed] [Google Scholar]
  13. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  15. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keskar S. S., Srinivasan M. C., Deshpande V. V. Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site. Biochem J. 1989 Jul 1;261(1):49–55. doi: 10.1042/bj2610049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Khasin A., Alchanati I., Shoham Y. Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl Environ Microbiol. 1993 Jun;59(6):1725–1730. doi: 10.1128/aem.59.6.1725-1730.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ko E. P., Akatsuka H., Moriyama H., Shinmyo A., Hata Y., Katsube Y., Urabe I., Okada H. Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus. Biochem J. 1992 Nov 15;288(Pt 1):117–121. doi: 10.1042/bj2880117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LEVY H. M., LEBER P. D., RYAN E. M. INACTIVATION OF MYOSIN BY 2,4-DINITROPHENOL AND PROTECTION BY ADENOSINE TRIPHOSPHATE AND OTHER PHOSPHATE COMPOUNDS. J Biol Chem. 1963 Nov;238:3654–3659. [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lee J. M., Hu Y., Zhu H., Cheng K. J., Krell P. J., Forsberg C. W. Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli. Can J Microbiol. 1993 Jan;39(1):134–139. doi: 10.1139/m93-020. [DOI] [PubMed] [Google Scholar]
  23. Lemesle-Varloot L., Henrissat B., Gaboriaud C., Bissery V., Morgat A., Mornon J. P. Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences. Biochimie. 1990 Aug;72(8):555–574. doi: 10.1016/0300-9084(90)90120-6. [DOI] [PubMed] [Google Scholar]
  24. Matte A., Forsberg C. W. Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85. Appl Environ Microbiol. 1992 Jan;58(1):157–168. doi: 10.1128/aem.58.1.157-168.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McDermid K. P., Forsberg C. W., MacKenzie C. R. Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85. Appl Environ Microbiol. 1990 Dec;56(12):3805–3810. doi: 10.1128/aem.56.12.3805-3810.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McGavin M., Forsberg C. W. Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85. J Bacteriol. 1988 Jul;170(7):2914–2922. doi: 10.1128/jb.170.7.2914-2922.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Paradis F. W., Zhu H., Krell P. J., Phillips J. P., Forsberg C. W. The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains. J Bacteriol. 1993 Dec;175(23):7666–7672. doi: 10.1128/jb.175.23.7666-7672.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sipat A., Taylor K. A., Lo R. Y., Forsberg C. W., Krell P. J. Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli. Appl Environ Microbiol. 1987 Mar;53(3):477–481. doi: 10.1128/aem.53.3.477-481.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith D. C., Forsberg C. W. alpha-Glucuronidase and Other Hemicellulase Activities of Fibrobacter succinogenes S85 Grown on Crystalline Cellulose or Ball-Milled Barley Straw. Appl Environ Microbiol. 1991 Dec;57(12):3552–3557. doi: 10.1128/aem.57.12.3552-3557.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thoma J. A., Rao G. V., Brothers C., Spradlin J., Li L. H. Subsite mapping of enzymes. Correlation of product patterns with Michaelis parameters and substrate-induced strain. J Biol Chem. 1971 Sep 25;246(18):5621–5635. [PubMed] [Google Scholar]
  31. Törrönen A., Kubicek C. P., Henrissat B. Amino acid sequence similarities between low molecular weight endo-1,4-beta-xylanases and family H cellulases revealed by clustering analysis. FEBS Lett. 1993 Apr 26;321(2-3):135–139. doi: 10.1016/0014-5793(93)80094-b. [DOI] [PubMed] [Google Scholar]

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