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Characterisation of Aspergillus niger prolyl aminopeptidase

Molecular Genetics and Genomics volume 272, pages 673–679 (2005)Cite this article

Abstract

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.

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References

  • Albertson NH, Koomey M (1993) Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae. Mol Microbiol 9:1203–1211

    Google Scholar 

  • Alonso J, Garcia JL (1996) Proline iminopeptidase gene from Xanthomonas campestris pv citri. Microbiology 142:2951–2957

    Google Scholar 

  • Altschul SF, Madden TL, Schaffer A, Zhang j, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    CAS  PubMed  Google Scholar 

  • Atlan D, Gilbert C, Blanc B, Portalier R (1994) Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp bulgaricus CNRZ 397. Microbiology 140:527–535

    Google Scholar 

  • Barret AJ, Rawlings ND, Woessner JF (1998) Handbook of proteolytic enzymes. Academic Press, San Diego

    Google Scholar 

  • Basten DEJW, Visser J, Schaap PJ (2001) Lysine aminopeptidase of Aspergillus niger. Microbiology 147:2045–2050

    Google Scholar 

  • Basten DEJW, Dekker PJT, Schaap PJ (2003) Aminopeptidase C of Aspergillus niger is a novel phenylalanine aminopeptidase. Appl Environ Microbiol 69:1246–1250

    Article  Google Scholar 

  • Bennet JW, Klich MA (1992) Aspergillus: biology and industrial applications. Butterworth-Heinemann, Boston

    Google Scholar 

  • Cunningham DF, O’Connor B (1997) Proline specific peptidases. Biochim Biophys Acta 1343:160–186

    Google Scholar 

  • Frey J, Röhm K-H (1979) External and internal forms of yeast aminopeptidase II. Eur J Biochem 97:169–173

    Google Scholar 

  • Gilbert C, Atlan D, Blanc B, Portalier R (1994) Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397: purification and characterization. Microbiology 140:537–542

    Google Scholar 

  • Goosen T, van Engelenburg F, Debets F, Swart K, Bos K, Van den Broek H (1989) Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis. Mol Gen Genet 219:282–288

    Article  Google Scholar 

  • Ito K, Inoue T, Kabashima T, Kanada M, Huang HS, Ma XH, Azmi N, Azab E, Yoshimoto T (2000) Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens. J Biochem 128:673–678

    Google Scholar 

  • Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403–405

    Article  CAS  PubMed  Google Scholar 

  • Kanatani A, Yoshimoto T, Kitazono A, Kokubo T, Tsuru D (1993) Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing and expression of the enzyme gene and characterization of the expressed enzyme. J Biochem 113:790–796

    Google Scholar 

  • Kitazono A, Yoshimoto T, Tsuru D (1992) Cloning, sequencing and high expression of the proline iminopeptidase gene from Bacillus coagulans. J Bacteriol 174:7919–7925

    Google Scholar 

  • Kitazono A, Ito K, Yoshimoto T (1994a) Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis. J Biochem 116:943–945

    Google Scholar 

  • Kitazono A, Kitano A, Tsuru D, Yoshimoto T (1994b) Isolation and characterization of the prolyl aminopeptidase gene (pap) from Aeromonas sobria: comparison with the Bacillus coagulans enzyme. J Biochem 116:818–825

    Google Scholar 

  • Kitazono A, Kitano A, Kabashima T, Ito K, Yoshimoto T (1996) Prolyl aminopeptidase is also present in Enterobacteriaceae: cloning and sequencing of the Hafnia alvei enzyme-gene and characterization of the expressed enzyme. J Biochem 119:468–474

    Google Scholar 

  • Kusters van Someren MA, Harmsen JAM, Kester HCM, Visser J (1991) Structure of the Aspergillus pelA gene and its expression in Aspergillus niger and Aspergillus nidulans. Curr Genet 20:293–299

    CAS  PubMed  Google Scholar 

  • Leenhouts K, Bolhuis A, Boot J, Deutz I, Toonen M, Venema G, Kok J, Ledeboer A (1998) Cloning, expression and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis. Appl Environ Microbiol 64:4736–4742

    Google Scholar 

  • Medrano FJ, Alonso J, Garcia JL, Romero A, Bode W, Gomis-Ruth FX (1998) Structure of proline iminopeptidase from Xanthomonas campestris pv citri: a prototype for the prolyl oligopeptidase family. EMBO J 17:1–9

    Google Scholar 

  • Mitta M, Miyagi M, Kato I, Tsunasawa S (1998) Identification of the catalytic triad residues of porcine liver acylamino acid-releasing enzyme. J Biochem 123:924–931

    Google Scholar 

  • Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358

    CAS  PubMed  Google Scholar 

  • Polgar L (1992) Structural relationship between lipases and peptidases of the prolyl oligopeptidase family. FEBS Lett 311:281–284

    Article  Google Scholar 

  • Pontecorvo G, Roper JA, Hemmons LJ, MacDonald KD, Bufton AWJ (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Troll W, Lindsley J (1955) A photometric method for the determination of proline. J Biol Chem 215:655–660

    Google Scholar 

  • Van den Hombergh JPT, van de Vondervoort PJI, Fraissinet-Tachet L, Visser J (1997) Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol 15:256–263

    Article  PubMed  Google Scholar 

  • Vanhoof G, Goossens F, Meester ID, Hendriks D, Scharpe S (1995) Proline motifs in peptides and their biological processing. FASEB J 9:736–744

    Google Scholar 

  • Yoshimoto T, Kabashima T, Uchikawa K, Inoue T, Tanaka N, Nakamura KT, Tsuru M, Ito K (1999) Crystal structure of prolyl aminopeptidase from Serratia marcescens. J Biochem 126:559–565

    Google Scholar 

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Acknowledgements

The authors thank H. Kester for assistance with the biochemical work. Daniëlle Basten was supported by DSM. P. Schaap was partially supported by a grant (WBI 4100) from the Dutch Technology Foundation (STW). All the work was carried out in compliance with Dutch and local laws governing genetic experimentation.

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Affiliations

  1. Section Fungal Genomics, Department of Microbiology, Wageningen University, Dreijenlaan 2, 6703, HA, Wageningen, The Netherlands

    Daniëlle E. J. W. Basten, Antoine P. H. A. Moers, Albert J. J. van. Ooyen & Peter J. Schaap

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  1. Daniëlle E. J. W. Basten
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  2. Antoine P. H. A. Moers
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  3. Albert J. J. van. Ooyen
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  4. Peter J. Schaap
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Corresponding author

Correspondence to Peter J. Schaap.

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Communicated by C.P. Hollenberg

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Basten, D.E.J.W., Moers, A.P.H.A., Ooyen, A.J.J.v. et al. Characterisation of Aspergillus niger prolyl aminopeptidase. Mol Genet Genomics 272, 673–679 (2005). https://doi.org/10.1007/s00438-004-1094-5

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  • DOI: https://doi.org/10.1007/s00438-004-1094-5

Keywords

  • Aspergillus niger
  • Prolyl aminopeptidase
  • Protease
  • Serine