Journal Article

Characterization of the novel Trypanosoma brucei inosine 5′-monophosphate dehydrogenase

NISHIMURA, SHIGENORI, SHINOHARA, TAKAHIRO, KUBATA, KILUNGA B., DJIKENG, APPOLINAIRE, BESSHO, TOMOAKI, SHUTO, SATOSHI, KUSUMOTO, TOSHIHIDE, NODA, MASANORI, MARTIN, SAMUEL K., MORII, SHOKO, INUI, TAKASHI, UCHIYAMA, SUSUMU, DUSZENKO, MICHAEL

2013

Overview

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 Km values of 30 and 1300 μm for IMP and NAD, respectively. The obtained Km 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 Ki values of 3·2 μm, 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.

• Publisher: Cambridge University Press
• Subject: African trypanosomiasis / Amino Acid Sequence / Animals / Binding Sites / Dehydrogenase / drug therapy / drugs / enzymes / Escherichia coli - genetics / Escherichia coli - metabolism / gel chromatography / guanine nucleotides / Humans / Hydrogen-Ion Concentration / IMP Dehydrogenase - antagonists & inhibitors / IMP Dehydrogenase - genetics / IMP Dehydrogenase - isolation & purification / IMP Dehydrogenase - metabolism / inosine monophosphate / Inosine Monophosphate - chemistry / Inosine Monophosphate - metabolism / Kinetics / Mammals / metabolism / Mycophenolic Acid - pharmacology / NAD (coenzyme) / NAD - metabolism / Nucleotides - pharmacology / Protein Multimerization / Recombinant Proteins / Ribonucleosides - pharmacology / Sequence Alignment / Sub-Saharan Africa / Trypanosoma brucei / Trypanosoma brucei brucei - enzymology / Trypanosoma brucei brucei - genetics / ultracentrifugation / Vector-borne diseases