IMP dehydrogenase from Pneumocystis carinii as a potential drug target

Mary Jeanne O'Gara, Chao Hung Lee, Geoffrey A. Weinberg, Jeniece M. Nott, Sherry F. Queener

Research output: Contribution to journalArticle

31 Scopus citations

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 he 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 Sφ1101). E. coli Sφ1101 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 K(m) for IMP of 21.7 ± 0.3 μM and an apparent K(m) for NAD of 314 ± 84 μM (mean ± standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 μM; n = 2).

Original languageEnglish (US)
Pages (from-to)40-48
Number of pages9
JournalAntimicrobial Agents and Chemotherapy
Volume41
Issue number1
DOIs
StatePublished - Jan 1997

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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