Overproduction, crystallization and preliminary X-ray diffraction analysis of a quinone oxidoreductase from Thermus thermophilus HB8.
A probable quinone oxidoreductase (MW = 32.1 kDa) from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography suggested the protein to be in a dimeric state. This protein enhanced the reduction activity of quinones by NADPH. It was crystallized in the absence and the presence of NADPH by the hanging-drop vapour-diffusion method. Both crystals were hexagonal, space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.6, c = 236.7 A for the apo form and a = b = 77.6, c = 235.9 A for the complex with NADPH. They diffract to better than 2.3 A resolution with synchrotron radiation. The asymmetric unit has one protein subunit (V(M) = 3.2 A(3) Da(-1) and V(sol) = 0.62 for the apo form), indicating that the twofold axis of the dimeric protein and the crystallographic twofold axis coincide.
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Yoshimitsu
Y
Shimomura
Kazuko
K
Sumiguchi-Agari
Ryoji
R
Masui
Seiki
S
Kuramitsu
Keiichi
K
Fukuyama
D001483:Base Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D021122:Protein Subunits; D011808:Quinone Reductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D021122:Protein Subunits; D011994:Recombinant Proteins; D011808:Quinone Reductases