Novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F). Cloning and expression of its gene in Escherichia coli.
A gene encoding a novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) was cloned, and its expression system was constructed in Escherichia coli. The 1.4-kilobase pair DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with KpnI and SmaI was found to express a protein binding FMN as a prosthetic group under control of the lac promoter in E. coli. This DNA fragment contained several putative open reading frames. The partial amino acid sequence of the polypeptide portion of the purified FMN-binding protein and its tryptic peptides were completely consistent with those deduced from the nucleotide sequence of the third open reading frame in the cloned SmaI-SmaI fragment of D. vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences. The nucleotide sequence of FMN-binding protein indicated that the protein is composed of 122 amino acids including an initiator Met residue and lacks a signal peptide for secretion. The main redox potential of the FMN-binding protein was measured as -325 mV using direct current cyclic and differential pulse voltammetric techniques and an electroreflectance method, suggesting that this FMN-binding protein functions as a redox protein like other FMN-binding proteins. Immunoblot analysis of the whole proteins from D. vulgaris (Miyazaki F) clearly indicated that this protein is expressed in this bacteria. However, the protein was found to have a primary structure distinct from those of other FMN-binding proteins and to be the smallest FMN-binding protein yet reported.
Kitamura
M
M
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Kojima
S
S
Ogasawara
K
K
Nakaya
T
T
Sagara
T
T
Niki
K
K
Miura
K
K
Akutsu
H
H
Kumagai
I
I
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D016969:Desulfovibrio vulgaris / Q000235:genetics* / Q000378:metabolism; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005420:Flavoproteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; C086046:FMN-binding protein, Desulfovibrio vulgaris; D005420:Flavoproteins; D005486:Flavin Mononucleotide