Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jul;177(13):3714–3720. doi: 10.1128/jb.177.13.3714-3720.1995

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis.

T K Wu 1, R W Busby 1, T A Houston 1, D B McIlwaine 1, L A Egan 1, C A Townsend 1
PMCID: PMC177087  PMID: 7601835

Abstract

Proclavaminate amidino hydrolase (PAH) catalyzes the reaction of guanidinoproclavaminic acid to proclavaminic acid and urea, a central step in the biosynthesis of the beta-lactamase inhibitor clavulanic acid. The gene encoding this enzyme (pah) was tentatively identified within the clavulanic acid biosynthetic cluster in Streptomyces clavuligerus by translation to a protein of the correct molecular mass (33 kDa) and appreciable sequence homology to agmatine ureohydrolase (M.B.W. Szumanski and S.M. Boyle, J. Bacteriol. 172:538-547, 1990) and several arginases, a correlation similarly recognized by Aidoo et al. (K. A. Aidoo, A. Wong, D. C. Alexander, R. A. R. Rittammer, and S. E. Jensen, Gene 147:41-46, 1994). Overexpression of the putative open reading frame as a 76-kDa fusion to the maltose-binding protein gave a protein having the catalytic activity sought. Cleavage of this protein with factor Xa gave PAH whose N terminus was slightly modified by the addition of four amino acids but exhibited unchanged substrate specificity and kinetic properties. Directly downstream of pah lies the gene encoding clavaminate synthase 2, an enzyme that carries out three distinct oxidative transformations in the in vivo formation of clavulanic acid. After the first of these oxidations, however, no further reaction was found to occur in vitro without the intervention of PAH. We have demonstrated that concurrent use of recombinant clavaminate synthase 2 and PAH results in the successful conversion of deoxyguanidinoproclavaminic acid to clavaminic acid, a four-step transformation. PAH has a divalent metal requirement, pH activity profile, and kinetic properties similar to those of other proteins of the broader arginase class.

Full Text

The Full Text of this article is available as a PDF (341.9 KB).

Selected References

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

  1. Aidoo K. A., Wong A., Alexander D. C., Rittammer R. A., Jensen S. E. Cloning, sequencing and disruption of a gene from Streptomyces clavuligerus involved in clavulanic acid biosynthesis. Gene. 1994 Sep 15;147(1):41–46. doi: 10.1016/0378-1119(94)90036-1. [DOI] [PubMed] [Google Scholar]
  2. Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Busby R. W., Chang M. D., Busby R. C., Wimp J., Townsend C. A. Expression and purification of two isozymes of clavaminate synthase and initial characterization of the iron binding site. General error analysis in polymerase chain reaction amplification. J Biol Chem. 1995 Mar 3;270(9):4262–4269. doi: 10.1074/jbc.270.9.4262. [DOI] [PubMed] [Google Scholar]
  5. Cavalli R. C., Burke C. J., Kawamoto S., Soprano D. R., Ash D. E. Mutagenesis of rat liver arginase expressed in Escherichia coli: role of conserved histidines. Biochemistry. 1994 Sep 6;33(35):10652–10657. doi: 10.1021/bi00201a012. [DOI] [PubMed] [Google Scholar]
  6. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  7. Cooper R. D. The enzymes involved in biosynthesis of penicillin and cephalosporin; their structure and function. Bioorg Med Chem. 1993 Jul;1(1):1–17. doi: 10.1016/s0968-0896(00)82098-2. [DOI] [PubMed] [Google Scholar]
  8. Jensen S. E., Wong A., Rollins M. J., Westlake D. W. Purification and partial characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus. J Bacteriol. 1990 Dec;172(12):7269–7271. doi: 10.1128/jb.172.12.7269-7271.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  10. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Marsh E. N., Chang M. D., Townsend C. A. Two isozymes of clavaminate synthase central to clavulanic acid formation: cloning and sequencing of both genes from Streptomyces clavuligerus. Biochemistry. 1992 Dec 22;31(50):12648–12657. doi: 10.1021/bi00165a015. [DOI] [PubMed] [Google Scholar]
  12. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  13. Ohtake A., Takiguchi M., Shigeto Y., Amaya Y., Kawamoto S., Mori M. Structural organization of the gene for rat liver-type arginase. J Biol Chem. 1988 Feb 15;263(5):2245–2249. [PubMed] [Google Scholar]
  14. Ouzounis C. A., Kyrpides N. C. On the evolution of arginases and related enzymes. J Mol Evol. 1994 Jul;39(1):101–104. doi: 10.1007/BF00178255. [DOI] [PubMed] [Google Scholar]
  15. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  16. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  17. Salowe S. P., Krol W. J., Iwata-Reuyl D., Townsend C. A. Elucidation of the order of oxidations and identification of an intermediate in the multistep clavaminate synthase reaction. Biochemistry. 1991 Feb 26;30(8):2281–2292. doi: 10.1021/bi00222a034. [DOI] [PubMed] [Google Scholar]
  18. Salowe S. P., Marsh E. N., Townsend C. A. Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: an unusual oxidative enzyme in natural product biosynthesis. Biochemistry. 1990 Jul 10;29(27):6499–6508. doi: 10.1021/bi00479a023. [DOI] [PubMed] [Google Scholar]
  19. Sandman K., Krzycki J. A., Dobrinski B., Lurz R., Reeve J. N. HMf, a DNA-binding protein isolated from the hyperthermophilic archaeon Methanothermus fervidus, is most closely related to histones. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5788–5791. doi: 10.1073/pnas.87.15.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Satishchandran C., Boyle S. M. Purification and properties of agmatine ureohydrolyase, a putrescine biosynthetic enzyme in Escherichia coli. J Bacteriol. 1986 Mar;165(3):843–848. doi: 10.1128/jb.165.3.843-848.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schrell A., Alt-Moerbe J., Lanz T., Schroeder J. Arginase of Agrobacterium Ti plasmid C58. DNA sequence, properties, and comparison with eucaryotic enzymes. Eur J Biochem. 1989 Oct 1;184(3):635–641. doi: 10.1111/j.1432-1033.1989.tb15060.x. [DOI] [PubMed] [Google Scholar]
  23. Schwacha A., Bender R. A. Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes. J Bacteriol. 1990 Sep;172(9):5477–5481. doi: 10.1128/jb.172.9.5477-5481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sumrada R. A., Cooper T. G. Nucleotide sequence of the Saccharomyces cerevisiae arginase gene (CAR1) and its transcription under various physiological conditions. J Bacteriol. 1984 Dec;160(3):1078–1087. doi: 10.1128/jb.160.3.1078-1087.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Szumanski M. B., Boyle S. M. Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli. J Bacteriol. 1990 Feb;172(2):538–547. doi: 10.1128/jb.172.2.538-547.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Takiguchi M., Haraguchi Y., Mori M. Human liver-type arginase gene: structure of the gene and analysis of the promoter region. Nucleic Acids Res. 1988 Sep 26;16(18):8789–8802. doi: 10.1093/nar/16.18.8789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ward J. M., Hodgson J. E. The biosynthetic genes for clavulanic acid and cephamycin production occur as a 'super-cluster' in three Streptomyces. FEMS Microbiol Lett. 1993 Jun 15;110(2):239–242. doi: 10.1111/j.1574-6968.1993.tb06326.x. [DOI] [PubMed] [Google Scholar]
  29. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  30. Winship P. R. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 1989 Feb 11;17(3):1266–1266. doi: 10.1093/nar/17.3.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wright F., Bibb M. J. Codon usage in the G+C-rich Streptomyces genome. Gene. 1992 Apr 1;113(1):55–65. doi: 10.1016/0378-1119(92)90669-g. [DOI] [PubMed] [Google Scholar]
  32. Xu Q., Baker B. S., Tata J. R. Developmental and hormonal regulation of the Xenopus liver-type arginase gene. Eur J Biochem. 1993 Feb 1;211(3):891–898. doi: 10.1111/j.1432-1033.1993.tb17622.x. [DOI] [PubMed] [Google Scholar]
  33. di Guan C., Li P., Riggs P. D., Inouye H. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene. 1988 Jul 15;67(1):21–30. doi: 10.1016/0378-1119(88)90004-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES