Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone.
Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.
Suzuki
Takashi
T
Department of Molecular Parasitology, Nagoya City University, Graduate School of Medical Sciences, Nagoya 467-8601, Japan. hakuna@med.nagoya-cu.ac.jp
Nihei
Coh-ichi
C
Yabu
Yoshisada
Y
Hashimoto
Tetsuo
T
Suzuki
Mitsuko
M
Yoshida
Ayako
A
Nagai
Kazuo
K
Hosokawa
Tomoyoshi
T
Minagawa
Nobuko
N
Suzuki
Shuichi
S
Kita
Kiyoshi
K
Ohta
Nobuo
N
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D003001:Cloning, Molecular; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012717:Sesquiterpenes / Q000494:pharmacology*; D014344:Trypanocidal Agents / Q000494:pharmacology*; D016689:Trypanosoma vivax / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000662:veterinary; D014354:Trypanosomiasis, Bovine / Q000469:parasitology; D000099188:Alternative Oxidase
D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D014344:Trypanocidal Agents; D000099188:Alternative Oxidase; C006640:ascofuranone