Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii. Expression and characterization of the recombinant protein.
The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.
Department of Biochemistry, University College Dublin, Belfield, Dublin 4, Ireland.
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O'Neill
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Mayhew
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Butler
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial; D044927:Electron-Transferring Flavoproteins; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D045854:Veillonellaceae / Q000235:genetics*
D004269:DNA, Bacterial; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D011994:Recombinant Proteins