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.
Shimomura
Yoshimitsu
Y
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Sumiguchi-Agari
Kazuko
K
Masui
Ryoji
R
Kuramitsu
Seiki
S
Fukuyama
Keiichi
K
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