title: The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of α- and γ-Linked Glutamic Acid Residues.
abstract: 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 (13)C-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 γ-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of α- and γ-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.
authors.affiliation: From the Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Jethro L
authors.initials: JL
authors.lastname: Hemmann
authors.affiliation: Institute of Pharmacology and Structural Biology, CNRS and Université de Toulouse-Paul Sabatier, 31077 Toulouse, France, and.
authors.forename: Olivier
authors.initials: O
authors.lastname: Saurel
authors.affiliation: From the Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Ochsner
authors.affiliation: Laboratoire des Interactions Plantes Microorganismes, INRA/CNRS 441-2594, Castanet Tolosan, France.
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Stodden
authors.affiliation: From the Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kiefer
authors.affiliation: Institute of Pharmacology and Structural Biology, CNRS and Université de Toulouse-Paul Sabatier, 31077 Toulouse, France, and.
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
authors.affiliation: From the Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland, jvorholt@ethz.ch.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D020601:Methylobacterium extorquens / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins
keywords: analytical chemistry; bacterial metabolism; biosynthesis; cell metabolism; mass spectrometry (MS); metabolomics; microbiology; nuclear magnetic resonance (NMR); one-carbon metabolism; polyglutamic acid