Characterization of the novel Trypanosoma brucei inosine 5'-monophosphate dehydrogenase.
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.
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Tomoaki
T
Bessho
Shoko
S
Morii
Toshihide
T
Kusumoto
Takahiro
T
Shinohara
Masanori
M
Noda
Susumu
S
Uchiyama
Satoshi
S
Shuto
Shigenori
S
Nishimura
Appolinaire
A
Djikeng
Michael
M
Duszenko
Samuel K
SK
Martin
Takashi
T
Inui
Kilunga B
KB
Kubata
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007291:Inosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009173:Mycophenolic Acid / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D055503:Protein Multimerization; D011994:Recombinant Proteins; D012263:Ribonucleosides / Q000494:pharmacology; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D009711:Nucleotides; D011994:Recombinant Proteins; D012263:Ribonucleosides; D009243:NAD; D007291:Inosine Monophosphate; C009174:ribavirin-5'-phosphate; C010052:mizoribine; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid