Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Feb 1;345(Pt 3):637–644.

pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger.

L Parenicová 1, J A Benen 1, H C Kester 1, J Visser 1
PMCID: PMC1220799  PMID: 10642523

Abstract

The nucleotide sequence data for pgaA and pgaB have been deposited with the EMBL, GenBank and DDBJ Databases under accession numbers Y18804 and Y18805 respectively. pgaA and pgaB, two genes encoding endopolygalacturonases (PGs, EC 3.2.1.15) A and B, were isolated from a phage genomic library of Aspergillus niger N400. The 1167 bp protein coding region of the pgaA gene is interrupted by one intron, whereas the 1234 bp coding region of the pgaB gene contains two introns. The corresponding proteins, PGA and PGB, consist of 370 and 362 amino acid residues respectively. Northern-blot analysis revealed that pgaA- and pgaB-specific mRNA accumulate in mycelia grown on sucrose. mRNAs are also present upon transfer to media containing D-galacturonic acid and pectin. Recombinant PGA and PGB were characterized with respect to pH optimum, activity on polygalacturonic acid, and mode of action and kinetics on oligogalacturonates of different chain length (n=3-7). At their pH optimum the specific activities in a standard assay for PGA (pH 4.2) and PGB (pH 5.0) were 16.5 mu+kat.mg(-1) and 8.3 mu+kat.mg(-1) respectively. Product progression analysis, using polygalacturonate as a substrate, revealed a random cleavage pattern for both enzymes and indicated processive behaviour for PGA. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 6. Using pectins of various degrees of methyl esterification, it was shown that PGA and PGB both preferred partially methylated substrates.

Full Text

The Full Text of this article is available as a PDF (210.1 KB).

Selected References

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

  1. Benen J. A., Kester H. C., Visser J. Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. Eur J Biochem. 1999 Feb;259(3):577–585. doi: 10.1046/j.1432-1327.1999.00080.x. [DOI] [PubMed] [Google Scholar]
  2. Bussink H. J., Brouwer K. B., de Graaff L. H., Kester H. C., Visser J. Identification and characterization of a second polygalacturonase gene of Aspergillus niger. Curr Genet. 1991 Sep;20(4):301–307. doi: 10.1007/BF00318519. [DOI] [PubMed] [Google Scholar]
  3. Bussink H. J., Buxton F. P., Fraaye B. A., de Graaff L. H., Visser J. The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes. Eur J Biochem. 1992 Aug 15;208(1):83–90. doi: 10.1111/j.1432-1033.1992.tb17161.x. [DOI] [PubMed] [Google Scholar]
  4. Bussink H. J., Buxton F. P., Visser J. Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II. Curr Genet. 1991 Jun;19(6):467–474. doi: 10.1007/BF00312738. [DOI] [PubMed] [Google Scholar]
  5. Bussink H. J., Kester H. C., Visser J. Molecular cloning, nucleotide sequence and expression of the gene encoding prepro-polygalacturonaseII of Aspergillus niger. FEBS Lett. 1990 Oct 29;273(1-2):127–130. doi: 10.1016/0014-5793(90)81066-w. [DOI] [PubMed] [Google Scholar]
  6. Carpita N. C., Gibeaut D. M. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 1993 Jan;3(1):1–30. doi: 10.1111/j.1365-313x.1993.tb00007.x. [DOI] [PubMed] [Google Scholar]
  7. Cary J. W., Brown R., Cleveland T. E., Whitehead M., Dean R. A. Cloning and characterization of a novel polygalacturonase-encoding gene from Aspergillus parasiticus. Gene. 1995 Feb 3;153(1):129–133. doi: 10.1016/0378-1119(94)00749-i. [DOI] [PubMed] [Google Scholar]
  8. Centis S., Guillas I., Séjalon N., Esuerré-Tugayé M. T. Endopolygalacturonase genes from Colletotrichum lindemuthianum: cloning of CLPG2 and comparison of its expression to that of CLPG1 during saprophytic and parasitic growth of the fungus. Mol Plant Microbe Interact. 1997 Aug;10(6):769–775. doi: 10.1094/MPMI.1997.10.6.769. [DOI] [PubMed] [Google Scholar]
  9. Cubero B., Scazzocchio C. Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. EMBO J. 1994 Jan 15;13(2):407–415. doi: 10.1002/j.1460-2075.1994.tb06275.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  12. Fraissinet-Tachet L., Reymond-Cotton P., Fèvre M. Characterization of a multigene family encoding an endopolygalacturonase in Sclerotinia sclerotiorum. Curr Genet. 1995 Dec;29(1):96–99. doi: 10.1007/BF00313199. [DOI] [PubMed] [Google Scholar]
  13. Goosen T., Bloemheuvel G., Gysler C., de Bie D. A., van den Broek H. W., Swart K. Transformation of Aspergillus niger using the homologous orotidine-5'-phosphate-decarboxylase gene. Curr Genet. 1987;11(6-7):499–503. doi: 10.1007/BF00384612. [DOI] [PubMed] [Google Scholar]
  14. Harmsen J. A., Kusters-van Someren M. A., Visser J. Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): indications of a pectin lyase gene family in A. niger. Curr Genet. 1990 Aug;18(2):161–166. doi: 10.1007/BF00312604. [DOI] [PubMed] [Google Scholar]
  15. Ho M. C., Whitehead M. P., Cleveland T. E., Dean R. A. Sequence analysis of the Aspergillus nidulans pectate lyase pelA gene and evidence for binding of promoter regions to CREA, a regulator of carbon catabolite repression. Curr Genet. 1995 Jan;27(2):142–149. doi: 10.1007/BF00313428. [DOI] [PubMed] [Google Scholar]
  16. Jeenes D. J., Marczinke B., MacKenzie D. A., Archer D. B. A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger. FEMS Microbiol Lett. 1993 Mar 1;107(2-3):267–271. doi: 10.1111/j.1574-6968.1993.tb06041.x. [DOI] [PubMed] [Google Scholar]
  17. Kato M., Sekine K., Tsukagoshi N. Sequence-specific binding sites in the Taka-amylase A G2 promoter for the CreA repressor mediating carbon catabolite repression. Biosci Biotechnol Biochem. 1996 Nov;60(11):1776–1779. doi: 10.1271/bbb.60.1776. [DOI] [PubMed] [Google Scholar]
  18. Kester H. C., Kusters-van Someren M. A., Müller Y., Visser J. Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis. Eur J Biochem. 1996 Sep 15;240(3):738–746. doi: 10.1111/j.1432-1033.1996.0738h.x. [DOI] [PubMed] [Google Scholar]
  19. Khanh N. Q., Albrecht H., Ruttkowski E., Löffler F., Gottschalk M., Jany K. D. Nucleotide and derived amino acid sequence of a pectinesterase cDNA isolated from Aspergillus niger strain RH 5344. Nucleic Acids Res. 1990 Jul 25;18(14):4262–4262. doi: 10.1093/nar/18.14.4262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kitamoto N., Kimura T., Kito Y., Ohmiya K., Tsukagoshi N. Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616. FEMS Microbiol Lett. 1993 Jul 15;111(1):37–41. doi: 10.1111/j.1574-6968.1993.tb06358.x. [DOI] [PubMed] [Google Scholar]
  21. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kusters-van Someren M. A., Harmsen J. A., Kester H. C., Visser J. Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans. Curr Genet. 1991 Sep;20(4):293–299. doi: 10.1007/BF00318518. [DOI] [PubMed] [Google Scholar]
  23. Kusters-van Someren M., Flipphi M., de Graaff L., van den Broeck H., Kester H., Hinnen A., Visser J. Characterization of the Aspergillus niger pelB gene: structure and regulation of expression. Mol Gen Genet. 1992 Jul;234(1):113–120. doi: 10.1007/BF00272352. [DOI] [PubMed] [Google Scholar]
  24. Martel M. B., Létoublon R., Fèvre M. Purification and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia sclerotiorum. FEMS Microbiol Lett. 1998 Jan 1;158(1):133–138. doi: 10.1111/j.1574-6968.1998.tb12812.x. [DOI] [PubMed] [Google Scholar]
  25. Melchers W. J., Verweij P. E., van den Hurk P., van Belkum A., De Pauw B. E., Hoogkamp-Korstanje J. A., Meis J. F. General primer-mediated PCR for detection of Aspergillus species. J Clin Microbiol. 1994 Jul;32(7):1710–1717. doi: 10.1128/jcm.32.7.1710-1717.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parenicová L., Benen J. A., Kester H. C., Visser J. pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. Eur J Biochem. 1998 Jan 15;251(1-2):72–80. doi: 10.1046/j.1432-1327.1998.2510072.x. [DOI] [PubMed] [Google Scholar]
  27. Robyt J. F., French D. Multiple attack and polarity of action of porcine pancreatic alpha-amylase. Arch Biochem Biophys. 1970 Jun;138(2):662–670. doi: 10.1016/0003-9861(70)90394-2. [DOI] [PubMed] [Google Scholar]
  28. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takashima S., Iikura H., Nakamura A., Masaki H., Uozumi T. Analysis of Cre1 binding sites in the Trichoderma reesei cbh1 upstream region. FEMS Microbiol Lett. 1996 Dec 15;145(3):361–366. doi: 10.1111/j.1574-6968.1996.tb08601.x. [DOI] [PubMed] [Google Scholar]
  30. VISHNIAC W., SANTER M. The thiobacilli. Bacteriol Rev. 1957 Sep;21(3):195–213. doi: 10.1128/br.21.3.195-213.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Whitehead M. P., Shieh M. T., Cleveland T. E., Cary J. W., Dean R. A. Isolation and characterization of polygalacturonase genes (pecA and pecB) from Aspergillus flavus. Appl Environ Microbiol. 1995 Sep;61(9):3316–3322. doi: 10.1128/aem.61.9.3316-3322.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Woodcock D. M., Crowther P. J., Doherty J., Jefferson S., DeCruz E., Noyer-Weidner M., Smith S. S., Michael M. Z., Graham M. W. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 1989 May 11;17(9):3469–3478. doi: 10.1093/nar/17.9.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wubben J. P., Mulder W., ten Have A., van Kan J. A., Visser J. Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea. Appl Environ Microbiol. 1999 Apr;65(4):1596–1602. doi: 10.1128/aem.65.4.1596-1602.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  35. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES