Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.
A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.
Kitamura
M
M
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Osaka, Japan.
Koshino
Y
Y
Kamikawa
Y
Y
Kohno
K
K
Kojima
S
S
Miura
K
K
Sagara
T
T
Akutsu
H
H
Kumagai
I
I
Nakaya
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016969:Desulfovibrio vulgaris / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007501:Iron / Q000032:analysis; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012416:Rubredoxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D015032:Zinc / Q000032:analysis
D005288:Ferredoxins; D011994:Recombinant Proteins; D012416:Rubredoxins; C066602:desulfoferrodoxin; D007501:Iron; C023091:rubredoxin-NAD+ reductase; D009247:NADH, NADPH Oxidoreductases; D015032:Zinc