Crystallization and preliminary crystallographic analysis of cyanide-insensitive alternative oxidase from Trypanosoma brucei brucei.
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 A resolution with 93.1% completeness and an overall R(merge) of 9.5%. The TAO crystals belonged to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 63.11, b = 136.44, c = 223.06 A. Assuming the presence of two rTAO molecules in the asymmetric unit (2 x 38 kDa), the calculated Matthews coefficient (V(M)) was 3.2 A(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.
Kido
Yasutoshi
Y
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Shiba
Tomoo
T
Inaoka
Daniel Ken
DK
Sakamoto
Kimitoshi
K
Nara
Takeshi
T
Aoki
Takashi
T
Honma
Teruki
T
Tanaka
Akiko
A
Inoue
Masayuki
M
Matsuoka
Shigeru
S
Moore
Anthony
A
Harada
Shigeharu
S
Kita
Kiyoshi
K
D003460:Crystallization; D018360:Crystallography, X-Ray; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D010088:Oxidoreductases; C020094:duroquinol oxidase