Characterization of the formyltransferase from Methylobacterium extorquensAM1

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methano genic and sulfate-reducing archaea. Here we describe the purification and c haracterization of formylmethanofuran-tetrahydromethanopterin formyltransfe rase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N-5-formylH(4)-MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activi ty with MFR and H4MPT isolated from the methanogenic archaeon Methano-therm obacter marburgensis (apparent K-m for formylMFR = 50 muM; apparent K-m for H4MPT = 30 muM). The enzyme is encoded by the ffsA gene and exhibits a seq uence identity of approximate to 40% with Ftr from methanogenic and sulfate -reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. I nterestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, t he respective polypeptides form a functional unit. Expression studies in Es cherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the comp lex showed sequence similarity to subunits of Fmd from methanogens, the com plex was not found to catalyze the oxidation of formylMFR. Detailed compari son of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site c ofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.