The one-carbon carrier methylofuran from Methylobacterium extorquens AM1 contains a large number of alpha- and gamma-linked glutamic acid residues
2016
Hemmann, Jethro L. | Saurel, Olivier | Ochsner, Andrea M. | Stodden, Barbara K. | Kiefer, Patrick | Milon, Alain | Vorholt, Julia A. | Institute of Microbiology | Institut de pharmacologie et de biologie structurale (IPBS) ; Université Toulouse III - Paul Sabatier (UT3) ; Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS) | Institut National de la Recherche Agronomique (INRA) | Ibisa; Region Midi-Pyrenees; European structural funds
International audience
Mostrar más [+] Menos [-]Inglés. Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a C-13-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely gamma-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of alpha- and gamma-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far.
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