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. 1997 Jan;41(1):40–48. doi: 10.1128/aac.41.1.40

IMP dehydrogenase from Pneumocystis carinii as a potential drug target.

M J O'Gara 1, C H Lee 1, G A Weinberg 1, J M Nott 1, S F Queener 1
PMCID: PMC163657  PMID: 8980752

Abstract

Mycophenolic acid, a specific inhibitor of IMP dehydrogenase (IMPDH; EC 1.1.1.205), is a potent inhibitor of Pneumocystis carinii growth in culture, suggesting that IMPDH may be a sensitive target for chemotherapy in this organism. The IMPDH gene was cloned as a first step to characterizing the enzyme and developing selective inhibitors. A 1.3-kb fragment containing a portion of the P. carinii IMPDH gene was amplified by PCR with two degenerate oligonucleotides based on conserved sequences in IMPDH from humans and four different microorganisms. Northern hybridization analysis showed the P. carinii IMPDH mRNA to be approximately 1.6 kb. The entire cDNA encoding P. carinii IMPDH was isolated and cloned. The deduced amino acid sequence of P. carinii IMPDH shared homology with bacterial (31 to 38%), protozoal (48 to 59%), mammalian (60 to 62%), and fungal (62%) IMPDH enzymes. The IMPDH cDNA was expressed by using a T7 expression system in an IMPDH-deficient strain of Escherichia coli (strain S phi 1101). E. coli S phi 1101 cells containing the P. carinii IMPDH gene were able to grow on medium lacking guanine, implying that the protein expressed in vivo was functional. Extracts of these E. coli cells contained IMPDH activity that had an apparent Km for IMP of 21.7 +/- 0.3 microM and an apparent Km for NAD of 314 +/- 84 microM (mean +/- standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 microM; n = 2).

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Selected References

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  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balzarini J., Karlsson A., Wang L., Bohman C., Horská K., Votruba I., Fridland A., Van Aerschot A., Herdewijn P., De Clercq E. Eicar (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide). A novel potent inhibitor of inosinate dehydrogenase activity and guanylate biosynthesis. J Biol Chem. 1993 Nov 25;268(33):24591–24598. [PubMed] [Google Scholar]
  3. Bartlett M. S., Edlind T. D., Durkin M. M., Shaw M. M., Queener S. F., Smith J. W. Antimicrotubule benzimidazoles inhibit in vitro growth of Pneumocystis carinii. Antimicrob Agents Chemother. 1992 Apr;36(4):779–782. doi: 10.1128/aac.36.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartlett M. S., Edlind T. D., Lee C. H., Dean R., Queener S. F., Shaw M. M., Smith J. W. Albendazole inhibits Pneumocystis carinii proliferation in inoculated immunosuppressed mice. Antimicrob Agents Chemother. 1994 Aug;38(8):1834–1837. doi: 10.1128/aac.38.8.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bartlett M. S., Fishman J. A., Queener S. F., Durkin M. M., Jay M. A., Smith J. W. New rat model of Pneumocystis carinii infection. J Clin Microbiol. 1988 Jun;26(6):1100–1102. doi: 10.1128/jcm.26.6.1100-1102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bartlett M. S., Queener S. F., Shaw M. M., Richardson J. D., Smith J. W. Pneumocystis carinii is resistant to imidazole antifungal agents. Antimicrob Agents Chemother. 1994 Aug;38(8):1859–1861. doi: 10.1128/aac.38.8.1859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bartlett M. S., Shaw M., Navaran P., Smith J. W., Queener S. F. Evaluation of potent inhibitors of dihydrofolate reductase in a culture model for growth of Pneumocystis carinii. Antimicrob Agents Chemother. 1995 Nov;39(11):2436–2441. doi: 10.1128/aac.39.11.2436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beck J. T., Zhao S., Wang C. C. Cloning, sequencing, and structural analysis of the DNA encoding inosine monophosphate dehydrogenase (EC 1.1.1.205) from Tritrichomonas foetus. Exp Parasitol. 1994 Feb;78(1):101–112. doi: 10.1006/expr.1994.1010. [DOI] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Collart F. R., Huberman E. Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs. J Biol Chem. 1988 Oct 25;263(30):15769–15772. [PubMed] [Google Scholar]
  11. Cushion M. T., Zhang J., Kaselis M., Giuntoli D., Stringer S. L., Stringer J. R. Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats. J Clin Microbiol. 1993 May;31(5):1217–1223. doi: 10.1128/jcm.31.5.1217-1223.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Edlind T. D., Bartlett M. S., Weinberg G. A., Prah G. N., Smith J. W. The beta-tubulin gene from rat and human isolates of Pneumocystis carinii. Mol Microbiol. 1992 Nov;6(22):3365–3373. doi: 10.1111/j.1365-2958.1992.tb02204.x. [DOI] [PubMed] [Google Scholar]
  13. Edman J. C., Edman U., Cao M., Lundgren B., Kovacs J. A., Santi D. V. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8625–8629. doi: 10.1073/pnas.86.22.8625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edman U., Edman J. C., Lundgren B., Santi D. V. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6503–6507. doi: 10.1073/pnas.86.17.6503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hedstrom L., Wang C. C. Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism. Biochemistry. 1990 Jan 30;29(4):849–854. doi: 10.1021/bi00456a001. [DOI] [PubMed] [Google Scholar]
  16. Hupe D. J., Azzolina B. A., Behrens N. D. IMP dehydrogenase from the intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophenolic acid. J Biol Chem. 1986 Jun 25;261(18):8363–8369. [PubMed] [Google Scholar]
  17. Kanzaki N., Miyagawa K. Nucleotide sequence of the Bacillus subtilis IMP dehydrogenase gene. Nucleic Acids Res. 1990 Nov 25;18(22):6710–6710. doi: 10.1093/nar/18.22.6710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krishnaiah K. V. Inosinic acid 5'-monophosphate dehydrogenase of Escherichia coli: purification by affinity chromatography and some properties. Arch Biochem Biophys. 1975 Oct;170(2):567–575. doi: 10.1016/0003-9861(75)90152-6. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Landman O., Shiffman D., Av-Gay Y., Aharonowitz Y., Cohen G. High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies. FEMS Microbiol Lett. 1991 Dec 1;68(3):239–244. doi: 10.1016/0378-1097(91)90362-e. [DOI] [PubMed] [Google Scholar]
  21. Lee C. H., Cheng C., Wang J., Lumeng L. Identification of hepatitis C viruses with a nonconserved sequence of the 5' untranslated region. J Clin Microbiol. 1992 Jun;30(6):1602–1604. doi: 10.1128/jcm.30.6.1602-1604.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee C. H., Lu J. J., Bartlett M. S., Durkin M. M., Liu T. H., Wang J., Jiang B., Smith J. W. Nucleotide sequence variation in Pneumocystis carinii strains that infect humans. J Clin Microbiol. 1993 Mar;31(3):754–757. doi: 10.1128/jcm.31.3.754-757.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu Y., Tidwell R. R., Leibowitz M. J. Inhibition of in vitro splicing of a group I intron of Pneumocystis carinii. J Eukaryot Microbiol. 1994 Jan-Feb;41(1):31–38. doi: 10.1111/j.1550-7408.1994.tb05931.x. [DOI] [PubMed] [Google Scholar]
  24. Natsumeda Y., Carr S. F. Human type I and II IMP dehydrogenases as drug targets. Ann N Y Acad Sci. 1993 Nov 30;696:88–93. doi: 10.1111/j.1749-6632.1993.tb17144.x. [DOI] [PubMed] [Google Scholar]
  25. Natsumeda Y., Ohno S., Kawasaki H., Konno Y., Weber G., Suzuki K. Two distinct cDNAs for human IMP dehydrogenase. J Biol Chem. 1990 Mar 25;265(9):5292–5295. [PubMed] [Google Scholar]
  26. Okada M., Shimura K., Shiraki H., Nakagawa H. IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells. J Biochem. 1983 Nov;94(5):1605–1613. [PubMed] [Google Scholar]
  27. Queener S. F. New drug developments for opportunistic infections in immunosuppressed patients: Pneumocystis carinii. J Med Chem. 1995 Nov 24;38(24):4739–4759. doi: 10.1021/jm00024a001. [DOI] [PubMed] [Google Scholar]
  28. Rave N., Crkvenjakov R., Boedtker H. Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 1979 Aug 10;6(11):3559–3567. doi: 10.1093/nar/6.11.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Sifri C. D., Wilson K., Smolik S., Forte M., Ullman B. Cloning and sequence analysis of a Drosophila melanogaster cDNA encoding IMP dehydrogenase. Biochim Biophys Acta. 1994 Jan 18;1217(1):103–106. [PubMed] [Google Scholar]
  31. Sinclair K., Wakefield A. E., Banerji S., Hopkin J. M. Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct. Mol Biochem Parasitol. 1991 Mar;45(1):183–184. doi: 10.1016/0166-6851(91)90042-5. [DOI] [PubMed] [Google Scholar]
  32. Sogin M. L., Edman J. C. A self-splicing intron in the small subunit rRNA gene of Pneumocystis carinii. Nucleic Acids Res. 1989 Jul 11;17(13):5349–5359. doi: 10.1093/nar/17.13.5349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  34. Stringer J. R., Stringer S. L., Zhang J., Baughman R., Smulian A. G., Cushion M. T. Molecular genetic distinction of Pneumocystis carinii from rats and humans. J Eukaryot Microbiol. 1993 Nov-Dec;40(6):733–741. doi: 10.1111/j.1550-7408.1993.tb04468.x. [DOI] [PubMed] [Google Scholar]
  35. Tiedeman A. A., Smith J. M. Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase. Gene. 1991 Jan 15;97(2):289–293. doi: 10.1016/0378-1119(91)90065-j. [DOI] [PubMed] [Google Scholar]
  36. Tiedeman A. A., Smith J. M. Nucleotide sequence of the guaB locus encoding IMP dehydrogenase of Escherichia coli K12. Nucleic Acids Res. 1985 Feb 25;13(4):1303–1316. doi: 10.1093/nar/13.4.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weinberg G. A., O'Gara M. J., Cushion M. T. Coinfection of rats with genetically diverse forms of Pneumocystis carinii demonstrated by P. carinii inosine monophosphate dehydrogenase gene polymorphism. J Eukaryot Microbiol. 1994 Sep-Oct;41(5):119S–119S. [PubMed] [Google Scholar]
  38. Wilson K., Collart F. R., Huberman E., Stringer J. R., Ullman B. Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani. J Biol Chem. 1991 Jan 25;266(3):1665–1671. [PubMed] [Google Scholar]
  39. Yamada Y., Natsumeda Y., Weber G. Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase. Biochemistry. 1988 Mar 22;27(6):2193–2196. doi: 10.1021/bi00406a057. [DOI] [PubMed] [Google Scholar]
  40. van Berg A. A., Mooyer P. A., van Lenthe H., Stet E. H., De Abreu R. A., van Kuilenburg A. B., van Gennip A. H. The IMP dehydrogenase inhibitor mycophenolic acid antagonizes the CTP synthetase inhibitor 3-deazauridine in MOLT-3 human leukemia cells: a central role for phosphoribosyl pyrophosphate. Biochem Pharmacol. 1995 Sep 28;50(7):1095–1098. doi: 10.1016/0006-2952(95)00228-r. [DOI] [PubMed] [Google Scholar]

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