CLONING, SEQUENCE-ANALYSIS, AND EXPRESSION OF THE GENES ENCODING THE 2 SUBUNITS OF THE METHYLOTROPHIC BACTERIUM W3A1 ELECTRON-TRANSFER FLAVOPROTEIN

The genes encoding the two different subunits of the electron transfer flavoprotein (ETF) from the methylotrophic bacterium W3A1 have been i dentified, cloned, and sequenced. A 0.8-kilobase pair DNA fragment was generated for use as a molecular probe by the amplification of genomi c sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the NH2-terminal amino acid sequenc es determined for the ETF subunits purified hom W3A1, The screening of a partial genomic minilibrary containing size-selected BamHI-SalI fra gments using this probe identified a 2.2-kilobase pair insert containi ng the complete coding sequences for both W3A1 ETF subunits, The genes are arranged in tandem in the genomic DNA with only 2 bases between t he TAG translation termination codon of the small subunit and the ATG translation initiation codon of the large subunit, The deduced amino a cid sequences of each of the W3A1 ETF subunits exhibit only similar to 30% identity with the corresponding subunits of the ETF from human, r at, and Paracoccus denitrificans, which as a group are greater than 50 % identical, Thus, the ETF from W3A1 may exhibit some unique structura l features that, like other differences in some of its physical and fu nctional properties, may distinguish this ETF from others in this fami ly. A highly homologous region near the COOH terminus of the large sub unit in all the ETF proteins was found to contain a sequence that matc hes in several ways the ADP-binding motif of flavoproteins and other d inucleotide-binding proteins, suggesting that the large subunit forms a portion of the FAD (or AMP) binding site in these proteins. Under co ntrol of the tac promoter, the cloned ETF subunit genes were co-expres sed in Escherichia coil producing the heterodimeric holoprotein with p hysical, spectral, and electron-accepting properties essentially ident ical to the ETF isolated from W3A1, The recombinant ETF serves as the electron acceptor for W3A1 trimethylamine dehydrogenase in vitro, accu mulating as the air-stable anionic semiquinone in the presence of exce ss trimethylamine, Fully reduced ETF could not be obtained even after prolonged enzymatic reduction.