1887

Abstract

A proline iminopeptidase gene () of an industrial strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or -specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50·7, 24·5 and 33·8 kDa proteins, respectively. The ORF3-encoded Pepl protein showed 65 % identity with the Pepl proteins from subsp. and subsp. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The gene was overexpressed in Purified Pepl hydrolysed only diand tripeptides with proline in the first position. Optimum Pepl activity was observed at pH 7·5 and 40 °C. A gel filtration analysis indicated that Pepl is a dimer of 53000. Pepl was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline--nitroanilide as substrate revealed and values of 0·8 mM and 350 mmol min mg, respectively, and a very high turnover number of 135000 s.

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1996-12-01
2022-01-17
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References

  1. 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]
  2. Baankreis R., Exterkate F. 1991; Characterization of a peptidase from Lactococcus lactis ssp. cremoris HP that hydrolyses di- and tripeptides containing proline or hydrophobic residues as the aminoterminal amino acid. Syst Appl Microbiol 14:317–323
    [Google Scholar]
  3. Buckel S., Bell A., Rao M., Hermodson M. 1986; An analysis of the structure of the product of the rbsA gene of Escherichia coliK12. J Biol Chem 261:7659–7662
    [Google Scholar]
  4. Chen J., Morrison D. 1987; Cloning of Streptococcus pneumoniaeDNA fragments in Escherichia coli requires vector protected by strong transcriptional terminators. Gene 55:179–187
    [Google Scholar]
  5. Doi E., Shibata D., Matoba T. 1981; Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem 118:173–184
    [Google Scholar]
  6. Dudley E., Steele J. 1994; Nucleotide sequence and distribution of the pepPN gene from Lactobacillus helveticus CNRZ32. FEMS Microbiol Lett 119:41–46
    [Google Scholar]
  7. El Soda M., Desmazeaud M. 1982; Les peptide hydrolases des lactobacilles du groupe Thermobacterium. I. Mise en évidence de ces activités chezLactobacillus helveticus, L. acidophilus, L. lactis et L. bulgaricus. . Can J Microbiol 28:1181–1188
    [Google Scholar]
  8. Evans I. J., Downie J. A. 1986; The nodI gene product of Rhiɀobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of thenodI and nodJgenes. Gene 45:95–101
    [Google Scholar]
  9. Fath M. J., Kolter R. 1993; ABC transporters: bacterial exporters. Microbiol Rev 57:995–1017
    [Google Scholar]
  10. Fox P. 1989; Proteolysis during cheese manufacture and ripening. J Dairy Sci 72:1379–1400
    [Google Scholar]
  11. Gilbert C., Atlan D., Blanc B., Portalier R. 1994; Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ397: purification and characterization. Microbiology 140:537–542
    [Google Scholar]
  12. Gilson E., Nikaido H., Hofnung M. 1982; Sequence of the malKgene in E. coli K12. Nucleic Acids Res 10:7449–7458
    [Google Scholar]
  13. Habibi-Najafi M., Lee B. 1995; Purification and characterization of proline iminopeptidase from Lactobacillus casei subsp.casei LLG. J Dairy Sci 78:251–259
    [Google Scholar]
  14. Hames B., Higgins S. 1985 Nucleic Acid Hybridisation: a Practical Approach. Oxford & Washington, DC: IRL Press.;
    [Google Scholar]
  15. Higgins C. 1992; ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8:67–113
    [Google Scholar]
  16. Hogg R., Voelker C., Voncarlowitz I. 1991; Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12. Mol Gen Genet 229:453–459
    [Google Scholar]
  17. Klein J., Schmidt U., Plapp R. 1994; Cloning, heterologous expression, and sequencing of a novel proline iminopeptidase gene, pepI, from Lactobacillus delbrueckiisubsp. lactis DSM7290. Micro-biology 140:1133–1139
    [Google Scholar]
  18. Kok J. 1990; Genetics of the proteolytic system of lactic acid bacteria. FEMS Microbiol Rev 87:15–42
    [Google Scholar]
  19. Kok J., de Vos W. 1994; The proteolytic system of lactic acid bacteria. In Genetics and Biotechnology of Lactic Acid Bacteria pp. 169–210 Gasson M., de Vos W. Edited by Glasgow: Blackie Academic & Professional;
    [Google Scholar]
  20. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
    [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  22. Makino K., Kim S., Shinagawa H., Amemura M., Nakata A. 1991; Molecular analysis of the cryptic and functionalphn operons for phosphate use in Escherichia coli K-12. J Bacteriol 173:2665–2672
    [Google Scholar]
  23. Mars I., Monnet V. 1995; An aminopeptidase P fromLactococcus lactis with original specificity. Biochim Biophys Acta 1243:209–215
    [Google Scholar]
  24. Meyer-Barton E. C., Klein J. R., Imam M., Plapp R. 1993; Cloning and sequence analysis of the X-propyl-dipeptidyl-amino- peptidase gene (pepX) from Lactobacillus delbrueckii spp spp. lactisDSM7290. Appl Microbiol Biotechnol 40:82–89
    [Google Scholar]
  25. Myöhônen S., Wahlfors J. 1993; Automated fluorescent primer extension. BioTechniques 14:16–17
    [Google Scholar]
  26. Palva A., Nyberg K., Palva I. 1988; Quantitation of α-amylase mRNA in Bacillus subtilis by nucleic acid sandwich hybridization. DNA 7:135–142
    [Google Scholar]
  27. Pritchard G., Coolbear T. 1993; The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol Rev 12:179–206
    [Google Scholar]
  28. Rawlings N., Polgar L., Barret A. 1991; A new family of serine type peptidases related to prolyl oligopeptidase. Biochem J 279:907–911
    [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
    [Google Scholar]
  31. Saurin W., Köster W., Dassa E. 1994; Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins. Mol Microbiol 12:993–1004
    [Google Scholar]
  32. Scripture J., Voelker C., Miller S., O’Donnell R., Polgar L., Rade J., Horazdovsky B., Hogg R. 1987; High-affinity l-arabinose transport operon nucleotide sequence and analysis of gene products. J Mol Biol 197:37–46
    [Google Scholar]
  33. Sofia H., Burland V., Daniels D., Plunkett G. III Blattner F. 1994; Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76·0 to 81·5 minutes. Nucleic Acids Res 22:2576–2586
    [Google Scholar]
  34. Stucky K., Klein J., Schüller A., Matern H., Henrich B., Plapp R. 1995; Cloning and DNA sequence analysis ofpepQ, a prolidase gene from Lactobacillus delbrueckii subsp.lactis DSM7290 and partial characterization of its product. Mol Gen Genet 247:494–500
    [Google Scholar]
  35. Tan P., Poolman B., Konings W. 1993; Proteolytic enzymes of Eactococcus lactis. J Dairy Res 60:269–286
    [Google Scholar]
  36. Troll W., Lindsley J. 1955; A photometric method for the determination of proline. J Biol Chem 215:655–661
    [Google Scholar]
  37. Tynkkynen S., Buist G., Kunji E., Kok J., Poolman B., Venema G., Haandrikman A. 1993; Genetic and biochemical charac-terization of the oligopeptide transport system of Lactococcus lactis. J Bacteriol 175:7523–7532
    [Google Scholar]
  38. Varmanen P., Steele J., Palva A. 1996; Characterization of a prolinase gene and its product and an adjacent ABC transporter gene from Lactobacillus helveticus. Microbiology 142:809–816
    [Google Scholar]
  39. Vesanto E., Varmanen P., Steele J. L., Palva A. 1994; Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase. Eur J Biochem 224:991–997
    [Google Scholar]
  40. Vesanto E., Savijoki K., Rantanen T., Steele J. L., Palva A. 1995; An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus. . Microbiology 141:3067–3075
    [Google Scholar]
  41. Vidgren G., Palva I., Pakkanen R., Lounatmaa K., Palva A. 1992; S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence. J Bacteriol 174:7419–7427
    [Google Scholar]
  42. Visser S. 1993; Proteolytic enzymes and their relation to cheese ripening and flavor: an overview. J Dairy Sci 76:329–350
    [Google Scholar]
  43. Walker J., Saraste M., Runswick M., Gay N. 1982; Distantly related sequences in the α and β subunits of ATP synthetase, myosin, kinases and other ATP requiring enzymes and a common nucleotide binding fold. EMBO J 1:945–951
    [Google Scholar]
  44. Williams S. G., Greenwood J. A., Jones C. W. 1992; Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter. Mol Microbiol 13:1755–1768
    [Google Scholar]
  45. Woodcock D., Crowther P., Doherty J., Jefferson S., De Cruz E., Noyer-Weidner M., Smith S., Michael M., Graham M. 1989; Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17:3469–3478
    [Google Scholar]
  46. Wu L., Welker N. E. 1991; Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription. J Bacteriol 173:4877–4888
    [Google Scholar]
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