Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens.
Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained. The flavodoxin holoprotein is over-expressed in E. coli from the cloned gene using its endogenous promoter.
Helms
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LR
Department of Biochemistry, Ohio State University, Columbus 43210.
Krey
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GD
Swenson
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RP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000096:biosynthesis; D003901:Desulfovibrio / Q000235:genetics*; D005418:Flavodoxin / Q000235:genetics*; D005420:Flavoproteins / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004269:DNA, Bacterial; D005418:Flavodoxin; D005420:Flavoproteins