ENERGY-TRANSDUCING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE - NUCLEOTIDE-SEQUENCES OF THE GENES AND PREDICTED AMINO-ACID-SEQUENCES OF THE SUBUNITS OF THE ENZYME FROM RHODOSPIRILLUM-RUBRUM

Based on the amino acid sequence of the N-terminus of the soluble subunit of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4 kbp was isolated from a genomic library of R. rubrum and sequenced. This sequence contained three open reading frames, constituting the genes nntA1, nntA2, and nntB of the R. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated alpha 1, alpha 2, and beta, respectively, and are considered to be the subunits of the R. rubrum transhydrogenase. The predicted amino acid sequence of the alpha 1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the alpha subunit of the Escherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a beta alpha beta fold in the corresponding region, and the purified, soluble alpha 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the beta subunit of the R. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the beta subunit of the E. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores of R. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the alpha 2 subunit of the R. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the alpha subunit of the E. coli transhydrogenases.