Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F).
The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = -434 and E2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.
Kitamura
M
M
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan.
Sagara
T
T
Taniguchi
M
M
Ashida
M
M
Ezoe
K
K
Kohno
K
K
Kojima
S
S
Ozawa
K
K
Akutsu
H
H
Kumagai
I
I
Nakaya
T
T
D001483:Base Sequence; D003001:Cloning, Molecular; D016969:Desulfovibrio vulgaris / Q000235:genetics*; D004563:Electrochemistry; D004566:Electrodes; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005418:Flavodoxin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005798:Genes, Bacterial; D006108:Graphite; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D005418:Flavodoxin; D011994:Recombinant Proteins; D006108:Graphite; D005486:Flavin Mononucleotide