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 identi fied by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase c hain 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 rev ealed that there are two possible dinucleotide-binding sites on the al pha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates tha t 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 recombina nt ETF was isolated from extracts of Escherichia coli. It is a heterod imer with subunits identical in size to the native protein. The isolat ed enzyme contains approximately I mol of FAD, but like the native pro tein it binds additional flavin to ave a total of about 2 mol of FAD/d imer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.