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. 1993 Nov;175(22):7290–7300. doi: 10.1128/jb.175.22.7290-7300.1993

dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

B Henrich 1, S Becker 1, U Schroeder 1, R Plapp 1
PMCID: PMC206872  PMID: 8226676

Abstract

Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties.

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Selected References

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

  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Bally M., Foglino M., Bruschi M., Murgier M., Lazdunski A. Nucleotide sequence of the promoter and amino-terminal encoding region of the Escherichia coli pepN gene. Eur J Biochem. 1986 Mar 17;155(3):565–569. doi: 10.1111/j.1432-1033.1986.tb09525.x. [DOI] [PubMed] [Google Scholar]
  3. Bally M., Murgier M., Lazdunski A. Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli. Mol Gen Genet. 1984;195(3):507–510. doi: 10.1007/BF00341454. [DOI] [PubMed] [Google Scholar]
  4. Becker S., Plapp R. Location of the dcp gene on the physical map of Escherichia coli. J Bacteriol. 1992 Mar;174(5):1698–1699. doi: 10.1128/jb.174.5.1698-1699.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Bolle P. A., Gilliquet V., Berben G., Dumont J., Hilger F. The complete sequence of K3B, a 7.9 kb fragment between PGK1 and CRY1 on chromosome III, reveals the presence of seven open reading frames. Yeast. 1992 Mar;8(3):205–213. doi: 10.1002/yea.320080306. [DOI] [PubMed] [Google Scholar]
  7. Burke J. F. High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates. Gene. 1984 Oct;30(1-3):63–68. doi: 10.1016/0378-1119(84)90105-7. [DOI] [PubMed] [Google Scholar]
  8. Bünning P., Holmquist B., Riordan J. F. Substrate specificity and kinetic characteristics of angiotensin converting enzyme. Biochemistry. 1983 Jan 4;22(1):103–110. doi: 10.1021/bi00270a015. [DOI] [PubMed] [Google Scholar]
  9. Carter J. R., Franden M. A., Aebersold R., McHenry C. S. Molecular cloning, sequencing, and overexpression of the structural gene encoding the delta subunit of Escherichia coli DNA polymerase III holoenzyme. J Bacteriol. 1992 Nov;174(21):7013–7025. doi: 10.1128/jb.174.21.7013-7025.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conlin C. A., Miller C. G. Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium. J Bacteriol. 1992 Mar;174(5):1631–1640. doi: 10.1128/jb.174.5.1631-1640.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Conlin C. A., Trun N. J., Silhavy T. J., Miller C. G. Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene. J Bacteriol. 1992 Sep;174(18):5881–5887. doi: 10.1128/jb.174.18.5881-5887.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cushman D. W., Cheung H. S., Sabo E. F., Ondetti M. A. Design of potent competitive inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino acids. Biochemistry. 1977 Dec 13;16(25):5484–5491. doi: 10.1021/bi00644a014. [DOI] [PubMed] [Google Scholar]
  13. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  15. Deutch C. E., Soffer R. L. Escherichia coli mutants defective in dipeptidyl carboxypeptidase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5998–6001. doi: 10.1073/pnas.75.12.5998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Doolittle R. F., Feng D. F., Johnson M. S., McClure M. A. Relationships of human protein sequences to those of other organisms. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):447–455. doi: 10.1101/sqb.1986.051.01.054. [DOI] [PubMed] [Google Scholar]
  17. Eckerskorn C., Mewes W., Goretzki H., Lottspeich F. A new siliconized-glass fiber as support for protein-chemical analysis of electroblotted proteins. Eur J Biochem. 1988 Oct 1;176(3):509–519. doi: 10.1111/j.1432-1033.1988.tb14308.x. [DOI] [PubMed] [Google Scholar]
  18. Eisenberg D., Weiss R. M., Terwilliger T. C. The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci U S A. 1984 Jan;81(1):140–144. doi: 10.1073/pnas.81.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Feng D. F., Doolittle R. F. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol. 1987;25(4):351–360. doi: 10.1007/BF02603120. [DOI] [PubMed] [Google Scholar]
  20. Foglino M., Gharbi S., Lazdunski A. Nucleotide sequence of the pepN gene encoding aminopeptidase N of Escherichia coli. Gene. 1986;49(3):303–309. doi: 10.1016/0378-1119(86)90366-5. [DOI] [PubMed] [Google Scholar]
  21. Gerendasy D., Ito J. Nucleotide sequence and transcription of the right early region of bacteriophage PRD1. J Bacteriol. 1990 Apr;172(4):1889–1898. doi: 10.1128/jb.172.4.1889-1898.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hamilton S., Miller C. G. Cloning and nucleotide sequence of the Salmonella typhimurium dcp gene encoding dipeptidyl carboxypeptidase. J Bacteriol. 1992 Mar;174(5):1626–1630. doi: 10.1128/jb.174.5.1626-1630.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Henrich B., Monnerjahn U., Plapp R. Peptidase D gene (pepD) of Escherichia coli K-12: nucleotide sequence, transcript mapping, and comparison with other peptidase genes. J Bacteriol. 1990 Aug;172(8):4641–4651. doi: 10.1128/jb.172.8.4641-4651.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Henrich B., Plapp R. Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector. Gene. 1986;42(3):345–349. doi: 10.1016/0378-1119(86)90239-8. [DOI] [PubMed] [Google Scholar]
  25. Henrich B., Schroeder U., Frank R. W., Plapp R. Accurate mapping of the Escherichia coli pepD gene by sequence analysis of its 5' flanking region. Mol Gen Genet. 1989 Feb;215(3):369–373. doi: 10.1007/BF00427031. [DOI] [PubMed] [Google Scholar]
  26. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol. 1981 Sep 25;151(3):389–409. doi: 10.1016/0022-2836(81)90003-6. [DOI] [PubMed] [Google Scholar]
  27. Jongeneel C. V., Bouvier J., Bairoch A. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett. 1989 Jan 2;242(2):211–214. doi: 10.1016/0014-5793(89)80471-5. [DOI] [PubMed] [Google Scholar]
  28. Klein J., Henrich B., Plapp R. Cloning and expression of the pepD gene of Escherichia coli. J Gen Microbiol. 1986 Aug;132(8):2337–2343. doi: 10.1099/00221287-132-8-2337. [DOI] [PubMed] [Google Scholar]
  29. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  30. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  33. Lazdunski A. M. Peptidases and proteases of Escherichia coli and Salmonella typhimurium. FEMS Microbiol Rev. 1989 Sep;5(3):265–276. doi: 10.1016/0168-6445(89)90035-1. [DOI] [PubMed] [Google Scholar]
  34. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Persson A. V., Russo S. F., Wilson I. B. A new chromogenic substrate for angiotensin-converting enzyme. Anal Biochem. 1978 Dec;91(2):674–683. doi: 10.1016/0003-2697(78)90554-7. [DOI] [PubMed] [Google Scholar]
  37. Persson A., Wilson E. B. A fluorogenic substrate for angiotensin-converting enzyme. Anal Biochem. 1977 Nov;83(1):296–303. doi: 10.1016/0003-2697(77)90538-3. [DOI] [PubMed] [Google Scholar]
  38. Pierotti A., Dong K. W., Glucksman M. J., Orlowski M., Roberts J. L. Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15. Biochemistry. 1990 Nov 13;29(45):10323–10329. doi: 10.1021/bi00497a006. [DOI] [PubMed] [Google Scholar]
  39. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Soffer R. L. Angiotensin-converting enzyme and the regulation of vasoactive peptides. Annu Rev Biochem. 1976;45:73–94. doi: 10.1146/annurev.bi.45.070176.000445. [DOI] [PubMed] [Google Scholar]
  43. Spector T. Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem. 1978 May;86(1):142–146. doi: 10.1016/0003-2697(78)90327-5. [DOI] [PubMed] [Google Scholar]
  44. Stirling C. J., Colloms S. D., Collins J. F., Szatmari G., Sherratt D. J. xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase. EMBO J. 1989 May;8(5):1623–1627. doi: 10.1002/j.1460-2075.1989.tb03547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  46. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sugiura M., Ito Y., Hirano K., Sawaki S. Detection of dipeptidase and tripeptidase and tripeptidase activities on polyacrylamide gel and cellulose acetate gel by the reduction of tetrazolium salts. Anal Biochem. 1977 Aug;81(2):481–484. doi: 10.1016/0003-2697(77)90724-2. [DOI] [PubMed] [Google Scholar]
  48. Vimr E. R., Green L., Miller C. G. Oligopeptidase-deficient mutants of Salmonella typhimurium. J Bacteriol. 1983 Mar;153(3):1259–1265. doi: 10.1128/jb.153.3.1259-1265.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vimr E. R., Miller C. G. Dipeptidyl carboxypeptidase-deficient mutants of Salmonella typhimurium. J Bacteriol. 1983 Mar;153(3):1252–1258. doi: 10.1128/jb.153.3.1252-1258.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yaron A. Dipeptidyl carboxypeptidase from Escherichia coli. Methods Enzymol. 1976;45:599–610. doi: 10.1016/s0076-6879(76)45053-x. [DOI] [PubMed] [Google Scholar]
  51. Yaron A., Mlynar D., Berger A. A dipeptidocarboxypeptidase from E. coli. Biochem Biophys Res Commun. 1972 May 26;47(4):897–902. doi: 10.1016/0006-291x(72)90577-3. [DOI] [PubMed] [Google Scholar]
  52. Yoshimoto T., Tone H., Honda T., Osatomi K., Kobayashi R., Tsuru D. Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101. J Biochem. 1989 Mar;105(3):412–416. doi: 10.1093/oxfordjournals.jbchem.a122678. [DOI] [PubMed] [Google Scholar]

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