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

摘要

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 Å resolution with 93.1% completeness and an overall R merge 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 Å. Assuming the presence of two rTAO molecules in the asymmetric unit (2 × 38 kDa), the calculated Matthews coefficient (V M) was 3.2 ų 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.