Advertisement

The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115)

  • Sun-Li ChongEmail author
  • Evy Battaglia
  • Pedro M. Coutinho
  • Bernard Henrissat
  • Maija Tenkanen
  • Ronald P. de VriesEmail author
Biotechnologically Relevant Enzymes and Proteins

Abstract

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)3-Xyl4, in the cultivation medium of the transformants. Expression analysis demonstrated that agu1 is not co-regulated with the predominant xylanase-encoding gene (xynA) of S. commune. The detailed sequence analysis of Agu1 demonstrated that this gene belongs to a novel glycoside hydrolase family (GH115) that also contains candidate genes from ascomycete fungi and bacteria. Phylogenetic analysis showed that the fungal GH115 α-glucuronidases are distinctly separate from the prokaryotic clade and distributed over three branches. The identification of putative genes encoding this enzyme in industrial fungi, such as Aspergillus oryzae and Hypocrea jecorina, will provide a starting point for further analysis of the importance of this enzyme for the hydrolysis of plant biomass.

Keywords

α-Glucuronidase Schizophyllum commune GH115 Gene expression Xylan degradation 

Notes

Acknowledgments

We thank Prof. Dr. Han Wösten for access to the S. commune genome sequence, Prof. Annele Hatakka (Department of Food and Environmental Sciences, University of Helsinki) for providing the S. commune growing facilities, Dr. Kristiina Mäkinen (Department of Food and Environmental Sciences, University of Helsinki) for providing the agu1 cDNA isolation facilities, Dr. Matti Siika-aho (VTT Technical Research Centre) and Dr. Sanna Koutaniemi (Department of Food and Environmental Sciences, University of Helsinki) for the purification of S. commune α-glucuronidase (for raising antibody for Western blotting), Dr. Luis G. Lugones (Microbiology, University of Utrecht) for useful suggestions for the growing of S. commune, and Dr. Karin Scholtmeijer (Microbiology, University of Utrecht) for the expression vector. The financial support from the Academy of Finland through the WoodWisdom-Net Programme (HemiPop project no. 1124281), Glycoscience Graduate School (S.-L. C.), and COST 928 and COST FP0602 Short Term Scientific Mission (STSM) are gratefully acknowledged. E. B. and R. P. V. were supported by the Dutch Technology Foundation STW, the applied science division of NWO, and the Technology Program of the Ministry of Economic Affairs (project no. 07063).

Supplementary material

253_2011_3157_MOESM1_ESM.doc (36 kb)
Supplemental Fig. 1 Nucleotide and translated amino acid sequences of S. commune agu1. Exons are shown in upper case letters and introns are shown in lower case letters. The signal peptide is highlighted and the N-glycosylation sites are underlined accordingly (DOC 36 kb)
253_2011_3157_MOESM2_ESM.doc (32 kb)
Supplemental Fig. 2 Alignment of Agu1, the N-terminal amino acid sequence of the purified α-glucuronidase from S. commune (Tenkanen and Siika-aho 2000), and the second GH115 member identified in the S. commune genome. Identical amino acids between the N-terminal sequence of the purified enzyme and Agu1 are underlined. Identical amino acids between Agu1 and the second GH115 member are indicated by stars. This alignment demonstrates that Agu1 encodes the purified enzyme and that the two GH115 members differ significantly from each other throughout the amino acid sequence (DOC 32 kb)
253_2011_3157_MOESM3_ESM.doc (198 kb)
Supplemental Fig. 3 Bootstrap-supported phylogenetic analysis of family GH115, showing all of the fungal proteins formed a group and separated from bacterial proteins. The characterized enzymes from P. stipitis (Scheffersomyces stipitis CBS 6054; Ryabova et al. 2009) and S. commune (this study) are indicated in bold type. Bootstrap values for the respective branches are shown (DOC 198 kb)
253_2011_3157_MOESM4_ESM.xls (28 kb)
Supplemental Table 1 Genes used in the alignment (XLS 28 kb)

References

  1. Aspinall GO (1980) Chemistry of cell wall polysaccharides. Biochem Plants 3:473–500Google Scholar
  2. Biely P, MacKenzie CR, Schneider H (1988) Production of acetyl xylan esterase by Trichoderma reesei and Schizophyllum commune. Can J Microbiol 34:767–772CrossRefGoogle Scholar
  3. Biely P, de Vries RP, Vrsanska M, Visser J (2000) Inverting character of α-glucuronidase A from Aspergillus tubingensis. Biochim Biophys Acta, Gen Subj 1474:360–364CrossRefGoogle Scholar
  4. Choi I, Kim H, Choi Y (2000) Gene cloning and characterization of α-glucuronidase of Bacillus stearothermophilus No. 236. Biosci Biotechnol Biochem 64:2530–2537CrossRefGoogle Scholar
  5. de Vries RP, Poulsen CH, Madrid S, Visser J (1998) aguA, the gene encoding an extracellular α-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid. J Bacteriol 180:243–249Google Scholar
  6. de Vries RP, Visser J, de Graaff LH (1999) CreA modulates the XlnR-induced expression on xylose of Aspergillus niger genes involved in xylan degradation. Res Microbiol 150:281–285CrossRefGoogle Scholar
  7. de Vries RP, van de Vondervoort PJI, Hendriks L, van de Belt M, Visser J (2002) Regulation of the α-glucuronidase-encoding gene (aguA) from Aspergillus niger. Mol Genet Genomics 268:96–102CrossRefGoogle Scholar
  8. Dons JJM, de Vries OMH, Wessels JGH (1979) Characterization of the genome of the basidiomycete Schizophyllum commune. Biochim Biophys Acta Nucleic Acids Protein Synth 563:100–112CrossRefGoogle Scholar
  9. Dudkin MS, Shkantova NG, Parfent'eva MA (1974) Structure of arabinoglucuronoxylan of Poa pratensis. Khim Prir Soedin 10:723–724Google Scholar
  10. Ebringerová A, Heinze T (2000) Xylan and xylan derivatives—biopolymers with valuable properties. 1. Naturally occurring xylans structures, isolation procedures and properties. Macromol Rapid Commun 21:542–556CrossRefGoogle Scholar
  11. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797Google Scholar
  12. Fauré R, Courtin CM, Delcour JA, Dumon C, Faulds CB, Fincher GB, Fort S, Fry SC, Halila S, Kabel MA, Pouvreau L, Quemener B, Rivet A, Saulnier L, Schols HA, Driquez H, O'Donohue MJ (2009) A brief and informationally rich naming system for oligosaccharide motifs of heteroxylans found in plant cell walls. Aust J Chem 62:1–5CrossRefGoogle Scholar
  13. Halgasova N, Kutejova E, Timko J (1994) Purification and some characteristics of the acetylxylan esterase from Schizophyllum commune. Biochem J 298:751–755Google Scholar
  14. Haltrich D, Steiner W (1994) Formation of xylanase by Schizophyllum commune: effect of medium components. Enzyme Microb Technol 16:229–235CrossRefGoogle Scholar
  15. Heneghan MN, McLoughlin L, Murray PG, Tuohy MG (2007) Cloning, characterization and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii. Enzyme Microb Technol 41:677–682CrossRefGoogle Scholar
  16. Huson DH, Richter DC, Rausch C, Dezulian T, Franz M, Rupp R (2007) Dendroscope: an interactive viewer for large phylogenetic trees. BMC Bioinformatics 8:460CrossRefGoogle Scholar
  17. Kolenová K, Vršanská M, Biely P (2005) Purification and characterization of two minor endo-β-1, 4-xylanases of Schizophyllum commune. Enzyme Microb Technol 36:903–910CrossRefGoogle Scholar
  18. Kolenová K, Vršanská M, Biely P (2006) Mode of action of endo-β-1, 4-xylanases of families 10 and 11 on acidic xylooligosaccharides. J Biotechnol 121:338–345CrossRefGoogle Scholar
  19. Kolenová K, Ryabova O, Vrsanska M, Biely P (2010) Inverting character of family GH115 α-glucuronidases. FEBS Lett 584:4063–4068CrossRefGoogle Scholar
  20. Lugones LG, Scholtmeijer K, Klootwijk R, Wessels JG (1999) Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol Microbiol 32:681–689 Google Scholar
  21. MacKenzie CR, Bilous D (1988) Ferulic acid esterase activity from Schizophyllum commune. Appl Environ Microbiol 54:1170–1173Google Scholar
  22. Niederpruem DJ, Hobbs H, Henry L (1964) Nutritional studies of development in Schizophyllum commune. J Bacteriol 88:1721–1729Google Scholar
  23. O'Connell KL, Stults JT (1997) Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymic digests. Electrophoresis 18:349–359CrossRefGoogle Scholar
  24. Ohm RA, de Jong JF, Lugones LG, Aerts A, Kothe E, Stajich JE, de Vries RP, Record E, Levasseur A, Baker SE, Bartholomew KA, Coutinho PM, Erdmann S, Fowler TJ, Gathman AC, Lombard V, Henrissat B, Knabe N, Kües U, Lilly WW, Lindquist E, Lucas S, Magnuson JK, Piumi F, Raudaskoski M, Salamov A, Schmutz J, Schwarze FWMR, van Kuyk PA, Horton JS, Grigoriev IV, Wösten HAB (2010) Genome sequence of the model mushroom Schizophyllum commune. Nat Biotechnol 28:957–963CrossRefGoogle Scholar
  25. Ruile P, Winterhalter C, Liebl W (1997) Isolation and analysis of a gene encoding α-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan. Mol Microbiol 23:267–279CrossRefGoogle Scholar
  26. Ryabova O, Vrsanska M, Kaneko S, van Zyl WH, Biely P (2009) A novel family of hemicellulolytic α-glucuronidase. FEBS Lett 583:1457–1462CrossRefGoogle Scholar
  27. Sambrook JF, Russell DW (2000) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  28. Schuren FH, Wessels JG (1994) Highly-efficient transformation of the homobasidiomycete Schizophyllum commune to phleomycin resistance. Curr Genet 26:179–183 Google Scholar
  29. Shao W, Obi SKC, Puls J, Wiegel J (1995) Purification and characterization of the α-glucuronidase from Thermoanaerobacterium sp. strain JW/SL-YS485, an important enzyme for the utilization of substituted xylans. Appl Environ Microbiol 61:1077–1081Google Scholar
  30. Siika-aho M, Tenkanen M, Buchert J, Puls J, Viikari L (1994) An α-glucuronidase from Trichoderma reesei Rut C-30. Enzyme Microb Technol 16:813–819CrossRefGoogle Scholar
  31. Teleman A, Lundqvist J, Tjerneld F, Stålbrand H, Dahlman O (2000) Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy. Carbohydr Res 329:807–815CrossRefGoogle Scholar
  32. Tenkanen M, Siika-aho M (2000) An α-glucuronidase of Schizophyllum commune acting on polymeric xylan. J Biotechnol 78:149–161CrossRefGoogle Scholar
  33. Tenkanen M, Luonteri E, Teleman A (1996) Effect of side groups on the action of β-xylosidase from Trichoderma reesei against substituted xylo-oligosaccharides. FEBS Lett 399:303–306CrossRefGoogle Scholar
  34. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  35. Timell TE (1967) Recent progress in the chemistry of wood hemicelluloses. Wood Sci Technol 1:45–70CrossRefGoogle Scholar
  36. Uchida H, Nanri T, Kawabata Y, Kusakabe I, Murakami K (1992) Purification and characterization of intracellular α-glucuronidase from Aspergillus niger 5-16. Biosci Biotechnol Biochem 56:1608–1615CrossRefGoogle Scholar
  37. van Peij NNME, Gielkens MMC, de Vries RP, Visser J, de Graaff LH (1998) The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl Environ Microbiol 64:3615–3619Google Scholar
  38. Verbruggen MA, Beldman G, Voragen AG (1998) Enzymic degradation of sorghum glucuronoarabinoxylans leading to tentative structures. Carbohydr Res 306:275–282CrossRefGoogle Scholar
  39. Yoshida S, Kusakabe I, Matsuo N, Shimizu K, Yasui T, Murakami K (1990) Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan. Agric Biol Chem 54:449–457Google Scholar
  40. Zhang Y, Frohman MA (1997) Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs. Methods Mol Biol 69:61–87Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Sun-Li Chong
    • 1
    Email author
  • Evy Battaglia
    • 2
  • Pedro M. Coutinho
    • 3
  • Bernard Henrissat
    • 3
  • Maija Tenkanen
    • 1
  • Ronald P. de Vries
    • 2
    • 4
    Email author
  1. 1.Department of Food and Environmental Sciences, Faculty of Agriculture and ForestryUniversity of HelsinkiHelsinkiFinland
  2. 2.Microbiology and Kluyver Centre for Industrial FermentationUtrecht UniversityUtrechtThe Netherlands
  3. 3.AFMB—UMR 6098, CNRSUniversités Aix-Marseille I and II, Case 932Marseille Cedex 9France
  4. 4.CBS-KNAW Fungal Biodiversity CentreUtrechtThe Netherlands

Personalised recommendations