1887

Abstract

Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the -nitroanilide (NA) peptides Suc-AAPF-NA, Suc-AAA-NA, K-NA, F-NA and P-NA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed prolyl aminopeptidase (PAP), displayed a 50-fold specificity for cleaving N-terminal Pro–X ( / =2.1×10 M s) compared with Ala–X or Val–X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 °C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. PAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an / hydrolase fold. Northern analysis indicated that mRNA levels were regulated by the composition of the growth medium. Highest transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus .

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2009-11-01
2019-11-16
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References

  1. Allen, P. J. & Emerson, R. ( 1949; ). Guayule rubber: microbiological improvement by shrub retting. Ind Eng Chem 41, 346–365.[CrossRef]
    [Google Scholar]
  2. Basten, D. E., Visser, J. & Schaap, P. J. ( 2001; ). Lysine aminopeptidase of Aspergillus niger. Microbiology 147, 2045–2050.
    [Google Scholar]
  3. Basten, D. E., Dekker, P. J. & Schaap, P. J. ( 2003; ). Aminopeptidase C of Aspergillus niger is a novel phenylalanine aminopeptidase. Appl Environ Microbiol 69, 1246–1250.[CrossRef]
    [Google Scholar]
  4. Basten, D. E., Moers, A. P., Ooyen, A. J. & Schaap, P. J. ( 2005; ). Characterisation of Aspergillus niger prolyl aminopeptidase. Mol Genet Genomics 272, 673–679.[CrossRef]
    [Google Scholar]
  5. Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. ( 2004; ). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783–795.[CrossRef]
    [Google Scholar]
  6. Bensadoun, A. & Weinstein, D. ( 1976; ). Assay of proteins in the presence of interfering materials. Anal Biochem 70, 241–250.[CrossRef]
    [Google Scholar]
  7. Bolumar, T., Sanz, Y., Aristoy, M. C. & Toldra, F. ( 2003; ). Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii. Appl Environ Microbiol 69, 227–232.[CrossRef]
    [Google Scholar]
  8. Cunningham, D. F. & O'Connor, B. ( 1997; ). Proline specific peptidases. Biochim Biophys Acta 1343, 160–186.[CrossRef]
    [Google Scholar]
  9. Doi, R. H. ( 2008; ). Cellulases of mesophilic microorganisms: cellulosome and noncellulosome producers. Ann N Y Acad Sci 1125, 267–279.[CrossRef]
    [Google Scholar]
  10. Felipe, M. S., Andrade, R. V., Arraes, F. B., Nicola, A. M., Maranhão, A. Q., Torres, F. A., Silva-Pereira, I., Poças-Fonseca, M. J., Campos, E. G. & other authors ( 2005; ). Transcriptional profiles of the human pathogenic fungus Paracoccidioides brasiliensis in mycelium and yeast cells. J Biol Chem 280, 24706–24714.[CrossRef]
    [Google Scholar]
  11. Fickers, P., Nicaud, J. M., Gaillardin, C., Destain, J. & Thonart, P. ( 2004; ). Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica. J Appl Microbiol 96, 742–749.[CrossRef]
    [Google Scholar]
  12. Fuke, Y. & Matsuoka, H. ( 1993; ). The purification and characterization of prolyl aminopeptidase from Penicillium camemberti. J Dairy Sci 76, 2478–2484.[CrossRef]
    [Google Scholar]
  13. Grassick, A., Murray, P. G., Thompson, R., Collins, C. M., Byrnes, L., Birrane, G., Higgins, T. M. & Tuohy, M. G. ( 2004; ). Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii. Eur J Biochem 271, 4495–4506.[CrossRef]
    [Google Scholar]
  14. Heneghan, M. N., McLoughlin, L., Murray, P. G. & Tuohy, M. G. ( 2007; ). Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii. Enzyme Microb Technol 41, 677–682.[CrossRef]
    [Google Scholar]
  15. Hiwatashi, K., Hori, K., Takahashi, K., Kagaya, A., Inoue, S., Sugiyama, T. & Takahashi, S. ( 2004; ). Purification and characterization of a novel prolyl aminopeptidase from Maitake (Grifola frondosa). Biosci Biotechnol Biochem 68, 1395–1397.[CrossRef]
    [Google Scholar]
  16. Inoue, T., Ito, K., Tozaka, T., Hatakeyama, S., Tanaka, N., Nakamura, K. T. & Yoshimoto, T. ( 2003; ). Novel inhibitor for prolyl aminopeptidase from Serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor. Arch Biochem Biophys 416, 147–154.[CrossRef]
    [Google Scholar]
  17. Ito, K., Inoue, T., Kabashima, T., Kanada, N., Huang, H. S., Ma, X., Azmi, N., Azab, E. & Yoshimoto, T. ( 2000; ). Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens. J Biochem 128, 673–678.[CrossRef]
    [Google Scholar]
  18. Jiménez, C. R., Huang, L., Qiu, Y. & Burlingame, A. L. ( 2001; ). In-gel digestion of proteins for MALDI-MS fingerprint mapping. Curr Protoc Protein Sci Chapter 16, Unit 16.4
    [Google Scholar]
  19. Kato, N., Murakoshi, Y., Kato, M., Kobayashi, T. & Tsukagoshi, N. ( 2002; ). Isomaltose formed by α-glucosidases triggers amylase induction in Aspergillus nidulans. Curr Genet 42, 43–50.[CrossRef]
    [Google Scholar]
  20. 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.[CrossRef]
    [Google Scholar]
  21. Kredics, L., Antal, Z., Szekeres, A., Hatvani, L., Manczinger, L., Vagvolgyi, C. & Nagy, E. ( 2005; ). Extracellular proteases of Trichoderma species. A review. Acta Microbiol Immunol Hung 52, 169–184.[CrossRef]
    [Google Scholar]
  22. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other authors ( 2007; ). clustal w and clustal_x version 2.0. Bioinformatics 23, 2947–2948.[CrossRef]
    [Google Scholar]
  23. McGuffin, L. J., Bryson, K. & Jones, D. T. ( 2000; ). The PSIPRED protein structure prediction server. Bioinformatics 16, 404–405.[CrossRef]
    [Google Scholar]
  24. Medrano, F. J., Alonso, J., Garcia, J. L., Romero, A., Bode, W. & Gomis-Ruth, F. X. ( 1998; ). Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family. EMBO J 17, 1–9.[CrossRef]
    [Google Scholar]
  25. Milewski, S., Mignini, F., Prasad, R. & Borowski, E. ( 2001; ). Unusual susceptibility of a multidrug-resistant yeast strain to peptidic antifungals. Antimicrob Agents Chemother 45, 223–228.[CrossRef]
    [Google Scholar]
  26. Morty, R. E., Shih, A. Y., Fulop, V. & Andrews, N. W. ( 2005; ). Identification of the reactive cysteine residues in oligopeptidase B from Trypanosoma brucei. FEBS Lett 579, 2191–2196.[CrossRef]
    [Google Scholar]
  27. Murray, P. G., Grassick, A., Laffey, C. D., Cuffe, M. M., Higgins, T., Savage, A. V., Planas, A. & Tuohy, M. G. ( 2001; ). Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3–1,4-beta-d-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70. Enzyme Microb Technol 29, 90–98.[CrossRef]
    [Google Scholar]
  28. Murray, P. G., Collins, C. M., Grassick, A. & Tuohy, M. G. ( 2003; ). Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product. Biochem Biophys Res Commun 301, 280–286.[CrossRef]
    [Google Scholar]
  29. O'Donoghue, A. J., Mahon, C. S., Goetz, D. H., O'Malley, J. M., Gallagher, D. M., Zhou, M., Murray, P. G., Craik, C. S. & Tuohy, M. G. ( 2008; ). Inhibition of a secreted glutamic peptidase prevents growth of the fungus Talaromyces emersonii. J Biol Chem 283, 29186–29195.[CrossRef]
    [Google Scholar]
  30. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I. & other authors ( 1992; ). The α/β hydrolase fold. Protein Eng 5, 197–211.[CrossRef]
    [Google Scholar]
  31. Polaina, J. & MacCabe, A. P. ( 2007; ). Industrial Enzymes: Structure, Function and Applications. Dordrecht: Springer.
  32. Polizeli, M. L., Rizzatti, A. C., Monti, R., Terenzi, H. F., Jorge, J. A. & Amorim, D. S. ( 2005; ). Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67, 577–591.[CrossRef]
    [Google Scholar]
  33. Raeder, U. & Broda, P. ( 1985; ). Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1, 17–20.[CrossRef]
    [Google Scholar]
  34. Reen, F. J., Murray, P. G. & Tuohy, M. G. ( 2003; ). Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding β-xylosidase from the thermophilic fungus Talaromyces emersonii. Biochem Biophys Res Commun 305, 579–585.[CrossRef]
    [Google Scholar]
  35. Stricker, A. R., Mach, R. L. & de Graaff, L. H. ( 2008; ). Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei). Appl Microbiol Biotechnol 78, 211–220.[CrossRef]
    [Google Scholar]
  36. Takagi, H. ( 2008; ). Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications. Appl Microbiol Biotechnol 81, 211–223.[CrossRef]
    [Google Scholar]
  37. Tuohy, M. G., Puls, J., Claeyssens, M., Vrsanska, M. & Coughlan, M. P. ( 1993; ). The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl β-d-xylosides and unsubstituted xylans. Biochem J 290, 515–523.
    [Google Scholar]
  38. Tuohy, M. G., Laffey, C. D. & Coughlan, M. P. ( 1994; ). Characterization of the individual components of the xylanolytic enzyme system of Talaromyces emersonii. Bioresour Technol 50, 37–42.[CrossRef]
    [Google Scholar]
  39. Tuohy, M. G., Walsh, D. J., Murray, P. G., Claeyssens, M., Cuffe, M. M., Savage, A. V. & Coughlan, M. P. ( 2002; ). Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochim Biophys Acta 1596, 366–380.[CrossRef]
    [Google Scholar]
  40. UniProt ( 2007; ). The Universal Protein Resource. Nucleic Acids Res 35, D193–D197.[CrossRef]
    [Google Scholar]
  41. Varmanen, P., Rantanen, T. & Palva, A. ( 1996; ). An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins. Microbiology 142, 3459–3468.[CrossRef]
    [Google Scholar]
  42. Yasothornsrikul, S. & Hook, V. Y. ( 2000; ). Detection of proteolytic activity by fluorescent zymogram in-gel assays. Biotechniques 28, 1166–1173.
    [Google Scholar]
  43. Yoshimoto, T., Kabashima, T., Uchikawa, K., Inoue, T., Tanaka, N., Nakamura, K. T., Tsuru, M. & Ito, K. ( 1999; ). Crystal structure of prolyl aminopeptidase from Serratia marcescens. J Biochem 126, 559–565.[CrossRef]
    [Google Scholar]
  44. Zhang, L., Jia, Y., Wang, L. & Fang, R. ( 2007; ). A proline iminopeptidase gene upregulated in planta by a LuxR homologue is essential for pathogenicity of Xanthomonas campestris pv. campestris. Mol Microbiol 65, 121–136.[CrossRef]
    [Google Scholar]
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