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IMP dehydrogenase from Pneumocystis carinii as a potential drug target
M J O'Gara1, C H Lee, G A Weinberg
1Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis 46202, USA.
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).
Insights
Mycophenolic acid inhibits Pneumocystis carinii growth by targeting inosine monophosphate dehydrogenase (IMPDH). Researchers cloned and expressed the P. carinii IMPDH gene, confirming its function and susceptibility to inhibition.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Mycophenolic acid is a specific inhibitor of inosine monophosphate dehydrogenase (IMPDH).
- IMPDH is a potential chemotherapeutic target for Pneumocystis carinii infections.
- Cloning the IMPDH gene is crucial for characterizing the enzyme and developing selective inhibitors.
Purpose of the Study:
- To clone and characterize the Pneumocystis carinii IMPDH gene.
- To express the P. carinii IMPDH enzyme in a heterologous system.
- To confirm the enzyme's functionality and susceptibility to mycophenolic acid.
Main Methods:
- Polymerase chain reaction (PCR) amplification using degenerate oligonucleotides.
- Northern hybridization to determine mRNA size.
- cDNA isolation, cloning, and sequencing.
- Heterologous expression in an IMPDH-deficient Escherichia coli strain.
- Enzyme activity assays and inhibition studies.
Main Results:
- A 1.3-kb fragment of the P. carinii IMPDH gene was amplified.
- The full-length P. carinii IMPDH cDNA was isolated and cloned.
- The deduced amino acid sequence showed homology to IMPDH enzymes from various organisms.
- Functional expression of P. carinii IMPDH in E. coli was confirmed by complementation.
- The expressed enzyme exhibited IMPDH activity inhibited by mycophenolic acid.
Conclusions:
- The P. carinii IMPDH gene was successfully cloned and expressed.
- The expressed P. carinii IMPDH enzyme is functional and sensitive to mycophenolic acid.
- These findings support IMPDH as a viable chemotherapeutic target against P. carinii.