1. Characterization of the novel Trypanosoma brucei inosine 5'-monophosphate dehydrogenase.
- Author
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Bessho T, Morii S, Kusumoto T, Shinohara T, Noda M, Uchiyama S, Shuto S, Nishimura S, Djikeng A, Duszenko M, Martin SK, Inui T, and Kubata KB
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Escherichia coli genetics, Escherichia coli metabolism, Humans, Hydrogen-Ion Concentration, IMP Dehydrogenase antagonists & inhibitors, IMP Dehydrogenase genetics, IMP Dehydrogenase metabolism, Inosine Monophosphate chemistry, Inosine Monophosphate metabolism, Kinetics, Mycophenolic Acid pharmacology, NAD metabolism, Nucleotides pharmacology, Protein Multimerization, Recombinant Proteins, Ribonucleosides pharmacology, Sequence Alignment, Trypanosoma brucei brucei genetics, IMP Dehydrogenase isolation & purification, Trypanosoma brucei brucei enzymology
- Abstract
There is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy. Trypanosoma lacks the enzymatic machinery for the de novo synthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5'-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinant Trypanosoma brucei IMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculated K m values of 30 and 1300 μ m for IMP and NAD, respectively. The obtained K m value of TbIMPDH for NAD is approximately 20-200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies show K i values of 3·2 μ m, 21 nM and 3·3 nM for ribavirin 5'-monophosphate, mycophenolic acid and mizoribine 5'-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.
- Published
- 2013
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