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Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei |
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| Authors: | Yasutoshi Kido Kosuke Nakamura Takashi Suzuki Hiroyuki Saimoto Daijiro Ohmori Shigeharu Harada |
| Affiliation: | a Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan b Department of Molecular Parasitology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan c Department of Materials Science, Faculty of Engineering, Tottori University, Tottori, Japan d Department of Chemistry, School of Medicine, Juntendo University, Tokyo, Japan e Biochemistry and Biomedical Sciences, School of Life Sciences, University of Sussex, Falmer, Brighton, UK f Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan |
| Abstract: | The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (Km of 338 μM and Vmax of 601 μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1. |
| Keywords: | AOX, alternative oxidase DM, n-dodecyl-β- smallcaps" >d-maltopyranoside EPR, electron paramagnetic resonance ICP-MS, inductively coupled plasma-mass spectrometer IPTG, isopropyl, β- smallcaps" >d-1-thiogalactoside kcat, molecular activity C10E8, octaethylene glycol-monododecylether OG, n-octyl-β- smallcaps" >d-glucopyranoside rTAO, recombinant trypanosome alternative oxidase SHAM, salycylhydroxamic acid TAO, trypanosome alternative oxidase Ubiquinol, reduced form ubiquinone |
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