Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Aug;175(16):4957–4961. doi: 10.1128/jb.175.16.4957-4961.1993

Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad.

M T Black 1
PMCID: PMC204959  PMID: 8394311

Abstract

Leader peptidase (LP) is the enzyme responsible for proteolytic cleavage of the amino acid leader sequence from bacterial preproteins. Recent data indicate that LP may be an unusual serine proteinase which operates without involvement of a histidine residue (M. T. Black, J. G. R. Munn, and A. E. Allsop, Biochem. J. 282:539-543, 1992; M. Sung and R. E. Dalbey, J. Biol. Chem. 267:13154-13159, 1992) and that, therefore, one or more alternative residues must perform the function of a catalytic base. With the aid of sequence alignments, site-specific mutagenesis of the gene encoding LP (lepB) from Escherichia coli has been employed to investigate the mechanism of action of the enzyme. Various mutant forms of plasmid-borne LP were tested for their abilities to complement the temperature-sensitive activity of LP in E. coli IT41. Data are presented which indicate that the only conserved amino acid residue possessing a side chain with the potential to ionize, and therefore with the potential to transfer protons, which cannot be substituted with a neutral side chain is lysine at position 145. The data suggest that the catalytic activity of LP is dependent on the operation of a serine-lysine catalytic dyad.

Full text

PDF
4957

Images in this article

Selected References

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

  1. Adachi H., Ohta T., Matsuzawa H. Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin. J Biol Chem. 1991 Feb 15;266(5):3186–3191. [PubMed] [Google Scholar]
  2. Baker R. K., Lively M. O. Purification and characterization of hen oviduct microsomal signal peptidase. Biochemistry. 1987 Dec 29;26(26):8561–8567. doi: 10.1021/bi00400a010. [DOI] [PubMed] [Google Scholar]
  3. Bilgin N., Lee J. I., Zhu H. Y., Dalbey R., von Heijne G. Mapping of catalytically important domains in Escherichia coli leader peptidase. EMBO J. 1990 Sep;9(9):2717–2722. doi: 10.1002/j.1460-2075.1990.tb07458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Black M. T., Munn J. G., Allsop A. E. On the catalytic mechanism of prokaryotic leader peptidase 1. Biochem J. 1992 Mar 1;282(Pt 2):539–543. doi: 10.1042/bj2820539. [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. CITRI N., GARBER N., SELA M. The effect of urea and guanidine hydrochloride on activity and optical rotation of penicillinase. J Biol Chem. 1960 Dec;235:3454–3459. [PubMed] [Google Scholar]
  7. Carter P., Wells J. A. Dissecting the catalytic triad of a serine protease. Nature. 1988 Apr 7;332(6164):564–568. doi: 10.1038/332564a0. [DOI] [PubMed] [Google Scholar]
  8. Dalbey R. E., Von Heijne G. Signal peptidases in prokaryotes and eukaryotes--a new protease family. Trends Biochem Sci. 1992 Nov;17(11):474–478. doi: 10.1016/0968-0004(92)90492-r. [DOI] [PubMed] [Google Scholar]
  9. Dalbey R. E., Wickner W. Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J Biol Chem. 1985 Dec 15;260(29):15925–15931. [PubMed] [Google Scholar]
  10. Dao-pin S., Anderson D. E., Baase W. A., Dahlquist F. W., Matthews B. W. Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. Biochemistry. 1991 Dec 10;30(49):11521–11529. doi: 10.1021/bi00113a006. [DOI] [PubMed] [Google Scholar]
  11. Dev I. K., Ray P. H., Novak P. Minimum substrate sequence for signal peptidase I of Escherichia coli. J Biol Chem. 1990 Nov 25;265(33):20069–20072. [PubMed] [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greenburg G., Shelness G. S., Blobel G. A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein. J Biol Chem. 1989 Sep 25;264(27):15762–15765. [PubMed] [Google Scholar]
  14. Herzberg O., Moult J. Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution. Science. 1987 May 8;236(4802):694–701. doi: 10.1126/science.3107125. [DOI] [PubMed] [Google Scholar]
  15. Higaki J. N., Evnin L. B., Craik C. S. Introduction of a cysteine protease active site into trypsin. Biochemistry. 1989 Nov 28;28(24):9256–9263. doi: 10.1021/bi00450a004. [DOI] [PubMed] [Google Scholar]
  16. Inada T., Court D. L., Ito K., Nakamura Y. Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli. J Bacteriol. 1989 Jan;171(1):585–587. doi: 10.1128/jb.171.1.585-587.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lamotte-Brasseur J., Dive G., Dideberg O., Charlier P., Frère J. M., Ghuysen J. M. Mechanism of acyl transfer by the class A serine beta-lactamase of Streptomyces albus G. Biochem J. 1991 Oct 1;279(Pt 1):213–221. doi: 10.1042/bj2790213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lin L. L., Little J. W. Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12. J Bacteriol. 1988 May;170(5):2163–2173. doi: 10.1128/jb.170.5.2163-2173.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Oefner C., D'Arcy A., Daly J. J., Gubernator K., Charnas R. L., Heinze I., Hubschwerlen C., Winkler F. K. Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. Nature. 1990 Jan 18;343(6255):284–288. doi: 10.1038/343284a0. [DOI] [PubMed] [Google Scholar]
  21. Ogawara H., Mantoku A., Shimada S. beta-lactamase from Streptomyces cacaoi. Purification and properties. J Biol Chem. 1981 Mar 25;256(6):2649–2655. [PubMed] [Google Scholar]
  22. Roland K. L., Little J. W. Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model. J Biol Chem. 1990 Aug 5;265(22):12828–12835. [PubMed] [Google Scholar]
  23. 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]
  24. Slilaty S. N., Little J. W. Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. Proc Natl Acad Sci U S A. 1987 Jun;84(12):3987–3991. doi: 10.1073/pnas.84.12.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Slilaty S. N., Vu H. K. The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad. Protein Eng. 1991 Dec;4(8):919–922. doi: 10.1093/protein/4.8.919. [DOI] [PubMed] [Google Scholar]
  26. Strynadka N. C., Adachi H., Jensen S. E., Johns K., Sielecki A., Betzel C., Sutoh K., James M. N. Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution. Nature. 1992 Oct 22;359(6397):700–705. doi: 10.1038/359700a0. [DOI] [PubMed] [Google Scholar]
  27. Sung M., Dalbey R. E. Identification of potential active-site residues in the Escherichia coli leader peptidase. J Biol Chem. 1992 Jul 5;267(19):13154–13159. [PubMed] [Google Scholar]
  28. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vandeyar M. A., Weiner M. P., Hutton C. J., Batt C. A. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene. 1988 May 15;65(1):129–133. doi: 10.1016/0378-1119(88)90425-8. [DOI] [PubMed] [Google Scholar]
  30. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  32. Wolfe P. B., Wickner W., Goodman J. M. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope. J Biol Chem. 1983 Oct 10;258(19):12073–12080. [PubMed] [Google Scholar]
  33. YaDeau J. T., Klein C., Blobel G. Yeast signal peptidase contains a glycoprotein and the Sec11 gene product. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):517–521. doi: 10.1073/pnas.88.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van Dijl J. M., de Jong A., Vehmaanperä J., Venema G., Bron S. Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. EMBO J. 1992 Aug;11(8):2819–2828. doi: 10.1002/j.1460-2075.1992.tb05349.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van Dijl J. M., van den Bergh R., Reversma T., Smith H., Bron S., Venema G. Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli. Mol Gen Genet. 1990 Sep;223(2):233–240. doi: 10.1007/BF00265059. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES