Structure of Methylofuran in Methylobacterium extorquens AM 1 1 The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM 1 Contains a Large Number of Alpha-and Gamma-linked Glutamic Acid Residues

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran (MFR) analog was proposed to be involved in formaldehyde oxidation in the Alphaproteobacterium. 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 MFR cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a 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 αand 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. INTRODUCTION First termed carbon dioxide reduction factor (1), methanofuran (MFR) was discovered in 1983 and its structure identified after purification from cell extracts of Methanobacterium thermoautotrophicum (2). MFR, tetrahydromethanopterin (H4MPT) (3,4) and coenzyme M (5) are cofactors that serve as carrier molecule for one-carbon units at the oxidation states of formate, formaldehyde and methanol, respectively, and were originally thought to be unique to the group of strict anaerobic methanogenic archaea (6). The importance of “archaeal” cofactors for one-carbon unit conversion was later extended to aerobic bacteria with the discovery of "methanogenic" enzymes in the methylotrophic model bacterium Methylobacterium extorquens AM1. This bacterium harbors enzymes with sequence identity to those from methanogens, and the enzymes were active with the cofactors MFR and H4MPT purified from M. thermoautotrophicum (7). While H4MPT was identified as dephospho-H4MPT (7), the identity of the assumed MFR analog remained unknown in M. http://www.jbc. org/cgi/doi/10.1074/jbc.M116.714741 The latest version is at JBC Papers in Press. Published on February 19, 2016 as Manuscript M116.714741 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 8, 2017 hp://w w w . jb.org/ D ow nladed from Structure of Methylofuran in Methylobacterium extorquens AM1 2 extorquens AM1 and other methylotrophic bacteria (8-10). In M. extorquens AM1, the one-carbon carrier coenzymes are involved in the oxidation of formaldehyde, which is generated from methanol (or methylamine) (11) (Fig. 1). After the enzymeassisted binding of formaldehyde to H4MPT (12), dehydrogenation (13) and hydrolysis to formylH4MPT (8), the one-carbon unit is transferred to the MFR-analog as shown with purified enzyme (14). Notably, and in contrast to the enzymes from methanogenic archaea, the formyl group is subsequently hydrolyzed and released as free formate by the formyltransferase/hydrolase complex (Fhc) (15). The oxidation pathway is then completed by formate dehydrogenases (16) or, alternatively, formate is fed into the serine cycle (17,18) for biomass formation via tetrahydrofolate (19). To date, five different variants of the MFR cofactor, labeled MFR-a to MFR-e, have been identified and structurally characterized in different methanogenic archaea (2,20-22). These MFR variants all have a common core structure consisting of an APMF-moiety (4-[[4-(2aminoethyl)phenoxy]-methyl]-2-furanmethanamine) linked to at least two γ-glutamic acid residues (Fig. 2A, example of MFR-a). The APMF core structure consists of a furan derivative attached to a tyramine residue. The different MFR variants show remarkable chemical variability, as different moieties, such as the hexanetetracarboxylic acid group in MFR-a or additional glutamic acid residues (MFR-b, MFR-d, MFR-e), can be attached to the side chain of the AMPF-γ-Glu2 structure. Modifications are also possible in the center of the polyglutamic acid side chain, as in the case of MFR-e, where an unusual spacer has been identified (22). In MFR-d and MFR-e, the number of glutamic acid residues is variable, ranging from 7-12 and 5-8, respectively (22). Some work has also been dedicated to the MFR biosynthesis, and four enzymes (MfnA-F) have been identified in Methanocaldococcus jannaschii so far (23-26). The essential role of enzymes that depend on the putative MFR analog in M. extorquens AM1 and other methylotrophs prompted us to identify the cofactor. We structurally characterize the cofactor from the model bacterium using high-resolution MS and NMR and propose the name "methylofuran" to take into account that it shows features not found in known MFR variants from methanogens so far. EXPERIMENTAL PROCEDURES