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Crystallization and preliminary crystallographic analysis of cyanide-insensitive alternative oxidase from Trypanosoma brucei brucei

journal contribution
posted on 2023-06-07, 20:46 authored by Yasutoshi Kido, Tomoo Shiba, Daniel Ken Inaoka, Kimitoshi Sakamoto, Takeshi Nara, Takashi Aoki, Teruki Honma, Akiko Tanaka, Masayuki Inoue, Shigeru Matsuoka, Anthony Moore, Shigeharu Haradaf, Kiyoshi Kita
Cyanide-insensitive alternative oxidase (AOX) is a mitochondrial membrane protein and a non-proton-pumping ubiquinol oxidase that catalyzes the four-electron reduction of dioxygen to water. In the African trypanosomes, trypanosome alternative oxidase (TAO) functions as a cytochrome-independent terminal oxidase that is essential for survival in the mammalian host; hence, the enzyme is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in haem-deficient Escherichia coli was purified and crystallized at 293 K by the hanging-drop vapour-diffusion method using polyethylene glycol 400 as a precipitant. X-ray diffraction data were collected at 100 K and were processed to 2.9 resolution with 93.1% completeness and an overall Rmerge of 9.5%. The TAO crystals belonged to the orthorhombic space group I222 or I212121, with unit-cell parameters a = 63.11, b = 136.44, c = 223.06 . Assuming the presence of two rTAO molecules in the asymmetric unit (2 38 kDa), the calculated Matthews coefficient (VM) was 3.2 3 Da 1, which corresponds to a solvent content of 61.0%. This is the first report of a crystal of the membrane-bound diiron proteins, which include AOXs.


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  • Published


Acta Crystallographica Section F: Structural Biology and Crystallization Communications




International Union of Crystallography





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  • Biochemistry Publications

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