Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Dec 1;272(2):369–376. doi: 10.1042/bj2720369

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

L E Kellett 1, D M Poole 1, L M Ferreira 1, A J Durrant 1, G P Hazlewood 1, H J Gilbert 1
PMCID: PMC1149709  PMID: 2125205

Abstract

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.

Full text

PDF
369

Images in this article

374Fig. 4.
on p.374
374Fig. 5.
on p.374

Selected References

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

  1. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dekker R. F., Richards G. N. Hemicellulases: their occurrence, purification, properties, and mode of action. Adv Carbohydr Chem Biochem. 1976;32:277–352. doi: 10.1016/s0065-2318(08)60339-x. [DOI] [PubMed] [Google Scholar]
  3. Ferreira L. M., Durrant A. J., Hall J., Hazlewood G. P., Gilbert H. J. Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase. Biochem J. 1990 Jul 1;269(1):261–264. doi: 10.1042/bj2690261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ghangas G. S., Wilson D. B. Cloning of the Thermomonospora fusca Endoglucanase E2 Gene in Streptomyces lividans: Affinity Purification and Functional Domains of the Cloned Gene Product. Appl Environ Microbiol. 1988 Oct;54(10):2521–2526. doi: 10.1128/aem.54.10.2521-2526.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gilbert H. J., Hall J., Hazlewood G. P., Ferreira L. M. The N-terminal region of an endoglucanase from Pseudomonas fluorescens subspecies cellulosa constitutes a cellulose-binding domain that is distinct from the catalytic centre. Mol Microbiol. 1990 May;4(5):759–767. doi: 10.1111/j.1365-2958.1990.tb00646.x. [DOI] [PubMed] [Google Scholar]
  6. Gilbert H. J., Sullivan D. A., Jenkins G., Kellett L. E., Minton N. P., Hall J. Molecular cloning of multiple xylanase genes from Pseudomonas fluorescens subsp. cellulosa. J Gen Microbiol. 1988 Dec;134(12):3239–3247. doi: 10.1099/00221287-134-12-3239. [DOI] [PubMed] [Google Scholar]
  7. Hall J., Gilbert H. J. The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa. Mol Gen Genet. 1988 Jul;213(1):112–117. doi: 10.1007/BF00333406. [DOI] [PubMed] [Google Scholar]
  8. Hall J., Hazlewood G. P., Barker P. J., Gilbert H. J. Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity. Gene. 1988 Sep 15;69(1):29–38. doi: 10.1016/0378-1119(88)90375-7. [DOI] [PubMed] [Google Scholar]
  9. Hall J., Hazlewood G. P., Huskisson N. S., Durrant A. J., Gilbert H. J. Conserved serine-rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing. Mol Microbiol. 1989 Sep;3(9):1211–1219. doi: 10.1111/j.1365-2958.1989.tb00271.x. [DOI] [PubMed] [Google Scholar]
  10. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  11. Hunkapiller M. W., Hewick R. M., Dreyer W. J., Hood L. E. High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol. 1983;91:399–413. doi: 10.1016/s0076-6879(83)91038-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. McGavin M., Forsberg C. W. Catalytic and substrate-binding domains of endoglucanase 2 from Bacteroides succinogenes. J Bacteriol. 1989 Jun;171(6):3310–3315. doi: 10.1128/jb.171.6.3310-3315.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Minton N. P., Atkinson T., Sherwood R. F. Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida. J Bacteriol. 1983 Dec;156(3):1222–1227. doi: 10.1128/jb.156.3.1222-1227.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Minton N. P., Bullman H. M., Scawen M. D., Atkinson T., Gilbert H. J. Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene. Gene. 1986;46(1):25–35. doi: 10.1016/0378-1119(86)90163-0. [DOI] [PubMed] [Google Scholar]
  17. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  19. Staden R. A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Res. 1980 Aug 25;8(16):3673–3694. doi: 10.1093/nar/8.16.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. West C. A., Elzanowski A., Yeh L. S., Barker W. C. Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases. FEMS Microbiol Lett. 1989 May;50(1-2):167–172. doi: 10.1016/0378-1097(89)90479-5. [DOI] [PubMed] [Google Scholar]
  22. Wong K. K., Tan L. U., Saddler J. N. Multiplicity of beta-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev. 1988 Sep;52(3):305–317. doi: 10.1128/mr.52.3.305-317.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES