The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of α- and γ-Linked Glutamic Acid Residues

Andrea M Ochsner,Barbara K Stodden,Alain Milon,Jethro L Hemmann,Olivier Saurel,Julia A Vorholt,Patrick Kiefer

doi:10.1074/jbc.m116.714741

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.

Highlights

First termed carbon dioxide reduction factor [1], methanofuran (MFR)2 was discovered in 1983, and its structure was identified after purification from cell extracts of Methanothermobacter thermautotrophicus [2]
Identification of Methylofuran and Characterization by NMR—The discovery of a formyltransferase-hydrolase complex (FhcABCD) in M. extorquens AM1 [14, 15] and its activity with MFR from M. marburgensis suggested the presence of an MFR analog in the aerobic methylotroph
In this work we demonstrate the presence of methylofuran, which is involved in methylotrophy, in M. extorquens AM1

Summary

Experimental Procedures

Chemicals—Formyl-MFR-a was purified from Methanothermobacter marburgensis as described [27] and was a gift from R.K. Purification of Methylofuran—For the purification of the cell extracts from batch A, an enzymatic assay was used for the identification of the fractions containing methylofuran This assay has been previously described as an alternative assay for the determination of formyltransferase activity [14]. Cell extracts from batch A were first purified on a Source 15Q column (16 ϫ 100 mm, Amersham Biosciences), equilibrated with 10 mM acetic acid at pH 5. Extraction of methylofuran was performed twice, to as described above, through the addition of 1 ml of water and 1.2 ml of methanol to the cell pellet and the boiling of the suspension at 100 °C for 5 min. The fractions containing methylofuran were measured by LC-MS for the analysis of the labeling pattern

Results

Measured Calculated Difference Molecular Retention

Furan moiety

Discussion