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. 1997 Feb;179(4):1193–1202. doi: 10.1128/jb.179.4.1193-1202.1997

Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1.

M Sugai 1, T Fujiwara 1, T Akiyama 1, M Ohara 1, H Komatsuzawa 1, S Inoue 1, H Suginaka 1
PMCID: PMC178816  PMID: 9023202

Abstract

A novel staphylolytic enzyme, ALE-1, acting on Staphylococcus aureus, was purified from a Staphylococcus capitis EPK1 culture supernatant. The optimal pH range for staphylolytic activity was 7 to 9. ALE-1 contains one Zn2+ atom per molecule. Analysis of peptidoglycan fragments released by ALE-1 indicated that the enzyme is a glycylglycine endopeptidase. The effects of various modulators were determined, and we found that o-phenanthroline, iodoacetic acid, diethylpyrocarbonate, and Cu2+ reduced the staphylolytic activity of ALE-1. beta-Casein, elastin, and pentaglycine were poor substrates for ALE-1. Molecular cloning data revealed that ALE-1 is composed of 362 amino acid residues and is synthesized as a precursor protein which is cleaved after Ala at position 35, thus producing a mature ALE-1 of 35.6 kDa. The primary structure of mature ALE-1 is very similar to the proenzyme form of lysostaphin. It has the modular design of an N-terminal domain of tandem repeats of a 13-amino-acid sequence fused to the active site containing C-terminal domain. Unlike lysostaphin, ALE-1 does not undergo processing of the N-terminal repeat domain in broth culture. ale-1 is encoded on the plasmid. Protein homology search suggested that ALE-1 and lysostaphin are members of the novel Zn2+ protease family with a homologous 38-amino-acid-long motif, Tyr-X-His-X(11)-Val-X(12/20)-Gly-X(5-6)-His.

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Selected References

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  1. Babenko Iu S., Kulikova T. Ia. Vzaimosviaz' mezhdu aktivnost'iu produtsenta liticheskikh fermentov Actinomyces recifensis var. lyticus 2435 i ego populiatsionnoi izmenchivost'iu. Antibiotiki. 1984 Jul;29(7):490–495. [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buist G., Kok J., Leenhouts K. J., Dabrowska M., Venema G., Haandrikman A. J. Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. J Bacteriol. 1995 Mar;177(6):1554–1563. doi: 10.1128/jb.177.6.1554-1563.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burke M. E., Pattee P. A. Purification and characterization of a staphylolytic enzyme from Pseudomonas aeruginosa. J Bacteriol. 1967 Mar;93(3):860–865. doi: 10.1128/jb.93.3.860-865.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carnicero A., Falcón M. A., Mansito T. B., Roldán J. M. A protease with staphylolytic activity from Pseudomonas aeruginosa PAKS I. FEMS Microbiol Lett. 1990 Jul;58(2):171–175. doi: 10.1111/j.1574-6968.1990.tb13973.x. [DOI] [PubMed] [Google Scholar]
  6. Carnicero A., Mansito T. B., Roldán J. M., Falcón M. A. Staphylolytic enzyme from Pseudomonas aeruginosa: characterization and immunocytochemical localization. Arch Microbiol. 1990;154(1):37–41. doi: 10.1007/BF00249175. [DOI] [PubMed] [Google Scholar]
  7. Chatterjee A. N., Wong W., Young F. E., Gilpin R. W. Isolation and characterization of a mutant of Staphylococcus aureus deficient in autolytic activity. J Bacteriol. 1976 Mar;125(3):961–967. doi: 10.1128/jb.125.3.961-967.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coleman J. E. Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem. 1992;61:897–946. doi: 10.1146/annurev.bi.61.070192.004341. [DOI] [PubMed] [Google Scholar]
  9. Coles N. W., Gilbo C. M., Broad A. J. Purification, properties and mechanism of action of a staphylolytic enzyme produced by Aeromonas hydrophila. Biochem J. 1969 Jan;111(1):7–15. doi: 10.1042/bj1110007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeHart H. P., Heath H. E., Heath L. S., LeBlanc P. A., Sloan G. L. The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus. Appl Environ Microbiol. 1995 Apr;61(4):1475–1479. doi: 10.1128/aem.61.4.1475-1479.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  12. Garcia J. L., Diaz E., Romero A., Garcia P. Carboxy-terminal deletion analysis of the major pneumococcal autolysin. J Bacteriol. 1994 Jul;176(13):4066–4072. doi: 10.1128/jb.176.13.4066-4072.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gustin J. K., Kessler E., Ohman D. E. A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion. J Bacteriol. 1996 Nov;178(22):6608–6617. doi: 10.1128/jb.178.22.6608-6617.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. HASH J. H. PURIFICATION AND PROPERTIES OF STAPHYLOLYTIC ENZYMES FROM CHALAROPSIS SP. Arch Biochem Biophys. 1963 Sep;102:379–388. doi: 10.1016/0003-9861(63)90245-5. [DOI] [PubMed] [Google Scholar]
  15. Heath H. E., Heath L. S., Nitterauer J. D., Rose K. E., Sloan G. L. Plasmid-encoded lysostaphin endopeptidase resistance of Staphylococcus simulans biovar staphylolyticus. Biochem Biophys Res Commun. 1989 May 15;160(3):1106–1109. doi: 10.1016/s0006-291x(89)80117-2. [DOI] [PubMed] [Google Scholar]
  16. Heinrich P., Rosenstein R., Böhmer M., Sonner P., Götz F. The molecular organization of the lysostaphin gene and its sequences repeated in tandem. Mol Gen Genet. 1987 Oct;209(3):563–569. doi: 10.1007/BF00331163. [DOI] [PubMed] [Google Scholar]
  17. Häse C. C., Finkelstein R. A. Bacterial extracellular zinc-containing metalloproteases. Microbiol Rev. 1993 Dec;57(4):823–837. doi: 10.1128/mr.57.4.823-837.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ichikawa J. K., Li C., Fu J., Clarke S. A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences. J Bacteriol. 1994 Mar;176(6):1630–1638. doi: 10.1128/jb.176.6.1630-1638.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iriarte M., Stainier I., Cornelis G. R. The rpoS gene from Yersinia enterocolitica and its influence on expression of virulence factors. Infect Immun. 1995 May;63(5):1840–1847. doi: 10.1128/iai.63.5.1840-1847.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iversen O. J., Grov A. Studies on lysostaphin. Separation and characterization of three enzymes. Eur J Biochem. 1973 Oct 5;38(2):293–300. doi: 10.1111/j.1432-1033.1973.tb03061.x. [DOI] [PubMed] [Google Scholar]
  21. Janda J. M., Reitano M., Bottone E. J. Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. J Clin Microbiol. 1984 Jan;19(1):44–47. doi: 10.1128/jcm.19.1.44-47.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Karow M., Georgopoulos C. Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB. J Bacteriol. 1992 Feb;174(3):702–710. doi: 10.1128/jb.174.3.702-710.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kessler E., Safrin M., Olson J. C., Ohman D. E. Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease. J Biol Chem. 1993 Apr 5;268(10):7503–7508. [PubMed] [Google Scholar]
  24. Kovach M. E., Hughes K. J., Everiss K. D., Peterson K. M. Identification of a ToxR-activated gene, tagE, that lies within the accessory colonization factor gene cluster of Vibrio cholerae O395. Gene. 1994 Oct 11;148(1):91–95. doi: 10.1016/0378-1119(94)90239-9. [DOI] [PubMed] [Google Scholar]
  25. Lange R., Hengge-Aronis R. The nlpD gene is located in an operon with rpoS on the Escherichia coli chromosome and encodes a novel lipoprotein with a potential function in cell wall formation. Mol Microbiol. 1994 Aug;13(4):733–743. doi: 10.1111/j.1365-2958.1994.tb00466.x. [DOI] [PubMed] [Google Scholar]
  26. Leclerc D., Asselin A. Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can J Microbiol. 1989 Aug;35(8):749–753. doi: 10.1139/m89-125. [DOI] [PubMed] [Google Scholar]
  27. Li S. L., Norioka S., Sakiyama F. Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus. J Bacteriol. 1990 Nov;172(11):6506–6511. doi: 10.1128/jb.172.11.6506-6511.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  29. Nakai K., Kidera A., Kanehisa M. Cluster analysis of amino acid indices for prediction of protein structure and function. Protein Eng. 1988 Jul;2(2):93–100. doi: 10.1093/protein/2.2.93. [DOI] [PubMed] [Google Scholar]
  30. Nord C. E., Sjöberg L., Wadström T., Wretlind B. Characterization of three Aeromonas and nine pseudomonas species by extracellular enzymes and haemolysins. Med Microbiol Immunol. 1975;161(2):79–87. doi: 10.1007/BF02121748. [DOI] [PubMed] [Google Scholar]
  31. Nord C. E., Wadström T., Dornbusch K., Wretlind B. Extracellular proteins in five clostridial species from human infections. Med Microbiol Immunol. 1975 Jul 2;161(3):145–154. doi: 10.1007/BF02121004. [DOI] [PubMed] [Google Scholar]
  32. Oshida T., Sugai M., Komatsuzawa H., Hong Y. M., Suginaka H., Tomasz A. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):285–289. doi: 10.1073/pnas.92.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Park P. W., Senior R. M., Griffin G. L., Broekelmann T. J., Mudd M. S., Mecham R. P. Binding and degradation of elastin by the staphylolytic enzyme lysostaphin. Int J Biochem Cell Biol. 1995 Feb;27(2):139–146. doi: 10.1016/1357-2725(94)00086-q. [DOI] [PubMed] [Google Scholar]
  34. Park S., Galloway D. R. Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa. Mol Microbiol. 1995 Apr;16(2):263–270. doi: 10.1111/j.1365-2958.1995.tb02298.x. [DOI] [PubMed] [Google Scholar]
  35. Recsei P. A., Gruss A. D., Novick R. P. Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1127–1131. doi: 10.1073/pnas.84.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robbe-Saule V., Coynault C., Norel F. The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses. FEMS Microbiol Lett. 1995 Feb 15;126(2):171–176. doi: 10.1111/j.1574-6968.1995.tb07412.x. [DOI] [PubMed] [Google Scholar]
  37. SCHINDLER C. A., SCHUHARDT V. T. LYSOSTAPHIN: A NEW BACTERIOLYTIC AGENT FOR THE STAPHYLOCOCCUS. Proc Natl Acad Sci U S A. 1964 Mar;51:414–421. doi: 10.1073/pnas.51.3.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. SCHINDLER C. A., SCHUHARDT V. T. PURIFICATION AND PROPERTIES OF LYSOSTAPHIN--A LYTIC AGENT FOR STAPHYLOCOCCUS AUREUS. Biochim Biophys Acta. 1965 Feb 15;97:242–250. doi: 10.1016/0304-4165(65)90088-7. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Schad P. A., Iglewski B. H. Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene. J Bacteriol. 1988 Jun;170(6):2784–2789. doi: 10.1128/jb.170.6.2784-2789.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  42. Smith T. J., Foster S. J. Characterization of the involvement of two compensatory autolysins in mother cell lysis during sporulation of Bacillus subtilis 168. J Bacteriol. 1995 Jul;177(13):3855–3862. doi: 10.1128/jb.177.13.3855-3862.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sugai M., Akiyama T., Komatsuzawa H., Miyake Y., Suginaka H. Characterization of sodium dodecyl sulfate-stable Staphylococcus aureus bacteriolytic enzymes by polyacrylamide gel electrophoresis. J Bacteriol. 1990 Nov;172(11):6494–6498. doi: 10.1128/jb.172.11.6494-6498.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sugai M., Koike H., Hong Y. M., Miyake Y., Nogami R., Suginaka H. Purification of a 51 kDa endo-beta-N-acetylglucosaminidase from Staphylococcus aureus. FEMS Microbiol Lett. 1989 Oct 15;52(3):267–272. doi: 10.1016/0378-1097(89)90209-7. [DOI] [PubMed] [Google Scholar]
  45. Sugai M., Komatsuzawa H., Akiyama T., Hong Y. M., Oshida T., Miyake Y., Yamaguchi T., Suginaka H. Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus. J Bacteriol. 1995 Mar;177(6):1491–1496. doi: 10.1128/jb.177.6.1491-1496.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sugai M., Komatsuzawa H., Ooku-Inomata K., Miyake Y., Ishida E., Suginaka H. Isolation and characterization of Staphylococcus aureus mutants which form altered cell clusters. Microbiol Immunol. 1994;38(12):995–999. doi: 10.1111/j.1348-0421.1994.tb02158.x. [DOI] [PubMed] [Google Scholar]
  47. Sugiyama H., Kashiba S., Amano T., Kotani S., Imanishi T. Purification and properties of a staphylolytic factor produced by a strain of Staphylococcus epidermidis. Biken J. 1967 Sep;10(3):109–120. [PubMed] [Google Scholar]
  48. Tanaka K., Takahashi H. Cloning, analysis and expression of an rpoS homologue gene from Pseudomonas aeruginosa PAO1. Gene. 1994 Dec 2;150(1):81–85. doi: 10.1016/0378-1119(94)90862-1. [DOI] [PubMed] [Google Scholar]
  49. Theisen M., Rioux C. R., Potter A. A. Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus. Infect Immun. 1993 May;61(5):1793–1798. doi: 10.1128/iai.61.5.1793-1798.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thompson J. S., Shockman G. D. A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls. Anal Biochem. 1968 Feb;22(2):260–268. doi: 10.1016/0003-2697(68)90315-1. [DOI] [PubMed] [Google Scholar]
  51. Trayer H. R., Buckley C. E., 3rd Molecular properties of lysostaphin, a bacteriolytic agent specific for Staphylococcus aureus. J Biol Chem. 1970 Sep 25;245(18):4842–4846. [PubMed] [Google Scholar]
  52. Vallee B. L., Auld D. S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry. 1990 Jun 19;29(24):5647–5659. doi: 10.1021/bi00476a001. [DOI] [PubMed] [Google Scholar]
  53. Wadstrom T., Vesterberg O. Studies on endo-beta-acetylglucosaminidase, staphylolytic peptidase, and N-acetylmuramyl-L-alanine amidase in lysostaphin and from Staphylococcus aureus. Acta Pathol Microbiol Scand B Microbiol Immunol. 1971;79(2):248–264. doi: 10.1111/j.1699-0463.1971.tb02152.x. [DOI] [PubMed] [Google Scholar]
  54. Wang X., Wilkinson B. J., Jayaswal R. K. Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. Gene. 1991 Jun 15;102(1):105–109. doi: 10.1016/0378-1119(91)90547-o. [DOI] [PubMed] [Google Scholar]
  55. Ward J. B., Perkins H. R. The purification and properties of two staphylolytic enzymes from Streptomyces griseus. Biochem J. 1968 Jan;106(1):69–76. doi: 10.1042/bj1060069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Warren R., Rogolsky M., Wiley B. B., Glasgow L. A. Effect of ethidium bromide on elimination of exfoliative toxin and bacteriocin production in Staphylococcus aureus. J Bacteriol. 1974 Jun;118(3):980–985. doi: 10.1128/jb.118.3.980-985.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Whitaker D. R. Lytic enzymes of Sorangium sp. Isolation and enzymatic properties of the alpha- and beta-lytic proteases. Can J Biochem. 1965 Dec;43(12):1935–1954. doi: 10.1139/o65-217. [DOI] [PubMed] [Google Scholar]
  58. Wren B. W. A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences. Mol Microbiol. 1991 Apr;5(4):797–803. doi: 10.1111/j.1365-2958.1991.tb00752.x. [DOI] [PubMed] [Google Scholar]
  59. Wretlind B., Hedén L., Wadström T. Formation of extracellular haemolysin by Aeromonas hydrophila in relation to protease and staphylolytic enzyme. J Gen Microbiol. 1973 Sep;78(1):57–65. doi: 10.1099/00221287-78-1-57. [DOI] [PubMed] [Google Scholar]
  60. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  61. Yoshimoto T., Tsuru D. Studies on bacteriolytic enzymes. II. Purification and some properties of two types of staphylolytic enzymes from Streptomyces griseus. J Biochem. 1972 Aug;72(2):379–390. doi: 10.1093/oxfordjournals.jbchem.a129913. [DOI] [PubMed] [Google Scholar]
  62. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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