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 ␥-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of ␣-and ␥-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.

SignificanceMethylotrophs play a crucial role in the global carbon cycle as they oxidize reduced one-carbon compounds such as methanol or methane to CO2. The step-wise conversion of these substrates generally takes place while the one-carbon units are bound to diffusible carrier molecules. However, our crystal structure of the formyltransferase/hydrolase complex from Methylorubrum extorquens demonstrates that the one-carbon carrier methylofuran tightly binds to the enzyme via its extended and branched polyglutamate chain. A swinging motion of the coenzyme then shuttles one-carbon units between the two active sites of the enzyme complex over a distance of 50 Å. The structure further highlights how the bacterial formate generation system relates to the archaeal CO2 fixation system.

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