Structure, Function, and Biosynthesis of the Coenzyme Methylofuran

Methylotrophy is a metabolic trait that allows certain organisms to grow on carbon substrates without any C-C bonds, such as methanol or methane. The conversion and oxidation of these compounds usually proceeds via the toxic intermediate formaldehyde and is assisted by coenzymes that act as carriers for one-carbon units. Most methylotrophic bacteria rely on the tetrahydromethanopterin(H4MPT)-linked pathway for the oxidation of formaldehyde. This pathway involves not only H4MPT but also requires an analog of methanofuran (MFR), a coenzyme originally thought to be unique to methanogenic archaea. Previous biochemical and genetic evidence suggested that the structure of bacterial MFR must be similar to the one from methanogens; however, its purification and structural analysis remained challenging. In this thesis, the isolation and structural elucidation of MFR from the well-studied methylotroph Methylorubrum extorquens is described. Using preparative chromatography combined with high-resolution mass spectrometry and NMR, the bacterial analog of MFR—which was termed methylofuran (MYFR)—was identified and characterized. The core structure of MYFR was found to be identical to archaeal MFR, except for the presence of a tyrosine instead of a tyramine residue. Surprisingly, an unprecedented polyglutamate side chain consisting of up to 24 glutamate residues was connected to the tyrosine residue. NMR analysis further revealed that the glutamates in MYFR showed both α- and γ-linkages. In the H4MPT-linked pathway, MYFR is required by the formyltransferase/hydrolase complex (Fhc), where the coenzyme functions as a carrier of formyl units. To investigate whether the unusually large polyglutamate side chain of MYFR plays a role in the interaction with Fhc, the structure of the enzyme-coenzyme complex was solved at 3.1 Å. Interestingly, MYFR is bound as a non-covalent prosthetic group and the polyglutamate side chain tightly interacts with a large patch of positively charged residues of Fhc. This binding site is centrally located between the two active sites for formyl transfer and hydrolysis, thus suggesting that the polyglutamate chain functions as a flexible linker that allows the formyl-carrying aminomethylfuran moiety to reach both active sites of the bifunctional enzyme complex. Formyl units can thus be efficiently shuttled between the two active sites, without the need for MYFR to dissociate from Fhc. The electron density of Fhc-bound MYFR additionally revealed that the polyglutamate side chain of MYFR is branched, i.e. some glutamates are involved in isopeptide bonds with other glutamates. The branched polyglutamate structure might be required to support the strong interaction with Fhc and seems to be a unique feature of MYFR that is not present in archaeal MFRs. Since the H4MPT-linked pathway is widespread in Bacteria, MYFR is expected to be present in many strains. To determine whether there is structural diversity of MYFR, a survey comprising 12 proteobacterial strains was performed. Only in two strains, MYFR in the form present in M. extorquens was found. In six strains, a second type of MYFR was discovered which contained a tyramine instead of the tyrosine residue. For four strains, no MYFR could be identified. Interestingly, the number of glutamates in MYFR was not conserved across strains. While some had similar numbers as found in M. extorquens (around 16–20), two strains contained MYFR with 12 or fewer glutamates. The complex structure of the polyglutamate side chain of MYFR posed the question of its biosynthetic origin. In Proteobacteria, many genes essential for H4MPT-linked methylotrophy have previously been identified. For several of them, the function remained unknown. To identify genes involved in MYFR biosynthesis, strains with deletions in three of these genes (orf5, orfY, and orf17) were analyzed. All three mutants were unable to produce functional MYFR; however, the Dorf5 strain was accumulating MYFR-Glu2, a short intermediate of MYFR. Overexpression of orf5 in M. extorquens led to a significant increase in the number of glutamates attached to MYFR, as up to 40 glutamates were detected. The enzyme was thus renamed to MyfA, highlighting that this is the first enzyme discovered to be specifically involved in MYFR biosynthesis. In vitro assays with purified MyfA revealed de novo polyglutamate synthesis activity using L/D-glutamate and L-glutamine as substrates. Unexpectedly, L-glutamine was found to be an essential component of the assay. Assays with labeled glutamine showed that glutamine was serving as a source of glutamyl units for the incorporation into polyglutamates. The incorporation presumably took place after conversion to glutamate, as MyfA also showed glutaminase activity. Additionally, MyfA was able to cleave short glutamate containing peptides, thus also acting as a peptidase. These findings trigger the question of how the in vitro activities relate to MYFR biosynthesis in vivo. Taken together, the results obtained in this thesis shed light on various aspects of the structure, function, and biosynthesis of MYFR. They provide an in-depth understanding of the role MYFR plays in theH4MPTlinked pathway, thus expanding our knowledge about the biochemical basis of methylotrophy. The complex structure of MYFR that was revealed in this thesis, combined with the enzymes involved in its biosynthesis, will provide exciting opportunities for future research

Methylofuran is a prosthetic group of the formyltransferase/hydrolase complex and shuttles one-carbon units between two active sites

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.ISSN:0027-8424ISSN:1091-649

Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain

Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography–mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.ISSN:0021-9258ISSN:1083-351

Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa-protein without functional annotation. The protein comprises two characteristic PepSY domains and we thus termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications towards the sustainable purification and separation of rare-earth elements.ISSN:0021-9258ISSN:1083-351

The one-carbon carrier methylofuran from Methylobacterium extorquens AM1 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 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