Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF

Metabolomics-driven discovery of the novel cofactor mycofactocin in mycobacteria revealed glycosylation with a cellulose-like sugar chain, regulation in response to ethanol and redox-activity.


Introduction
Coenzymes are small molecules indispensable for the catalytic activity of many enzymes. While coenzymes like NAD + or FAD are ubiquitous in nature and are essential for the core metabolism of all forms of life, specialized cofactors like pyrroloquinoline quinone (PQQ) 1 and coenzyme F 420 2 are restricted to certain microbial phyla, but typically involved in extraordinary metabolic processes like methylotrophy, methanogenesis, or detoxication processes. Moreover, specialized cofactors serve as model systems for the evolution of cofactors and their co-evolution with their associated enzyme families. They can be regarded as examples of low-molecular weight natural products that modify, extend or enhance microbial metabolism. Mycobacteria are particularly rich in unusual redox cofactors and antioxidants that contribute to redox balance and metabolic plasticity. For instance, mycothiol 3,4 or ergothioneine 5 protect Mycobacterium tuberculosis from oxidative stress and support detoxication pathways. Coenzyme F 420 is involved, e.g., in cell wall biosynthesis 6 or defense against nitrosative stress in mycobacteria. 7 Moreover, some antimycobacterial drugs like pretomanid 8 are administered as prodrugs and will only develop bioactivity aer biotransformation by a coenzyme F 420 -dependent reductase. 9 Mycofactocin (MFT) is a putative redox-cofactor whose existence has been postulated on the basis of comparative genomics and bioinformatics. 10,11 Its molecular identity and structure, however, have remained elusive to date. The MFT biosynthetic gene cluster is highly conserved and widespread among mycobacteria. The inactivation of the MFT gene locus in the model species Mycolicibacterium smegmatis (synonym: Mycobacterium smegmatis) as well as M. tuberculosis resulted in the inability of the mutants to utilize ethanol as a sole source of carbon and further disturbances of mycobacterial redox homeostasis were revealed. 12 Involvement of MFT in methanol metabolism was reported as well. 13 These recent results strongly support the hypothesis that MFT is a redox cofactor and might represent a tness factor of mycobacteria during some stages of infection.
The architecture of the MFT gene cluster (Fig. 1A) suggested that the resulting natural product is a ribosomally synthesized and post-translationally modied peptide (RiPP). 14 Several in vitro studies have contributed to a preliminary biosynthetic model of MFT (Fig. 1B): the precursor peptide MA of M. smegmatis consisting of 31 amino acids is produced by the ribosome and bound by its chaperone MB. Subsequently, the terminal core peptide consisting of Val and Tyr is oxidatively decarboxylated and cyclized by the radical SAM enzyme MC. [15][16][17] The resulting cyclic core structure is released by the peptidase ME 18 forming 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP). 19 Just recently, it was shown that MD, an enzyme homologous to the L-lactate dehydrogenase LldD2 20 catalyzes the oxidative deamination of AHDP to yield pre-mycofactocin (PMFT). 21 The same study demonstrated by voltammetry that the a-keto amide moiety of PMFT is redox-active and can be reduced to PMFTH 2 (midpoint potential: À255 mV). Efficient reduction was also achieved by the action of carveol dehydrogenase using carveol as an electron donor in vitro. 21 Therefore, PMFT likely represents the redoxcenter of MFT, as riboavin is the redox-active core of FMN and FAD.
Although these current hypotheses are plausible, all these known metabolic intermediates have only been observed in vitro and could therefore represent artifacts, making the verication of their relevance in vivo urgently desired. Furthermore, additional steps of MFT biosynthesis, the function of the mF gene as well as the chemical structure of natural MFT awaited experimental clarication. In this study, we conrm the current biosynthetic model of MFT in vivo, detected several novel oligoglycosylated MFT congeners and elucidated their structure. We show that MFTs are decorated with a b-1,4-glucan chain and provide genetic evidence that glycosylation is performed by the glycosyltransferase MF. Finally, we show dependence of MFT formation on ethanol and corroborate its cofactor function by activity-based metabolic proling.

Discovery of mycofactocins by metabolomics
In order to identify potential mycofactocin congeners in mycobacteria, we used the fast-growing and weakly pathogenic species M. smegmatis MC 2 155 as a model organism and developed a metabolomics approach combining metabolic induction and labeling to specically trace MFT congeners. Assuming that MFT production would be stimulated by alcohols, we cultivated bacteria in media containing 10 g L À1 ethanol. Furthermore, we used stable isotope labeling to obtain candidate molecules compatible with the proposed biosynthetic pathway: since the C-terminal core peptide of MA is composed of Val and Tyr, we reasoned that MFT congeners could be specically labeled by feeding L-Val-13 C 5 and L-Tyr-13 C 9 . Intracellular contents were extracted and analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC-MS). Compounds were detected by in silico grouping of coeluting isotopic peaks and adducts (feature nding). Aerwards, 13 C-labeled compounds were deduced computationally (Data Set 1 †). According to the established biosynthetic pathway we expected 13 carbons to remain 13 C-labeled aer oxidative decarboxylation of the Val-Tyr core peptide. We therefore searched for compounds that displayed an exchange of 13 carbons, resulting in a mass shi of +13.04362 Da (ESI Fig. S1 †). This approach revealed a list of only twelve candidate compounds. Strikingly, the exact mass and proposed sum formula of three of these compounds corresponded to known intermediates of MFT, recently described in vitro, namely AHDP, PMFT as well as PMFTH 2 . In addition to these compounds, several labeled molecules with increasing molecular weight were detected. Some co-eluting candidates with a mass difference of +17.02654 could be explained as NH 4 + adducts of each other. The remaining nine candidate compounds (Table 1) were grouped based on their chromatographic retention times, eluting closely to either PMFT or PMFTH 2 (approx. 7.2 min and 6.9 min, respectively). Intriguingly, members of the two groups could be arranged in pairs with a mass difference of two hydrogen atoms leading us to assume that each group represented derivatives of either PMFT or PMFTH 2 . Thus, we termed these two clusters of molecules mycofactocinones (MFT) and mycofactocinols (MFTH 2 ), respectively.
Notably, some mycofactocinols eluted as two chromatographically separated isomers. For instance, the dominant PMFTH 2 eluted at 6.8 min, while the minor isomer eluted at 6.5 min. These two compounds displayed highly similar MS/MS spectra (ESI Fig. S2 †) and most likely represent tautomeric forms. For reasons of simplicity, only the more prevalent isomer was considered during metabolomics studies. We then performed MS/MS networking ( Fig. 2), an approach that clusters compounds based on similarity of their MS/MS fragmentation pattern and therefore potentially related chemical scaffolds. 22 Interestingly, candidates retrieved from 13 C-labeling experiments clustered with further putative MFT congeners. The mass difference between the rst candidate mycofactocinol (MFT-1H 2 ) with an exact mass of 397.17395 Da and PMFTH 2 was +162.05303 Da, which corresponded to a hexose sugar.
Furthermore, the MS/MS spectrum of MFT-1H 2 (Fig. 2) showed a fragment ion that corresponded to the mass of the putative aglycon (m/z 236.13 [M + H] + ), thus supporting the assumption that MFT-1H 2 was a glycosylated derivative of PMFTH 2 . MS/MS networking also revealed a recurrent mass difference of 14.01565 between compounds, indicating that methylation might occur as well. We thus assumed that the MFT candidate molecules could be explained as glycosylated or glycosylated and monomethylated species of PMFT(H 2 ). In analogy to coenzyme F 420 -n, where n indicates the number of glutamyl residues in the side chain, 2 we named the glycosylated molecules MFT-n(H 2 ) with n representing the number of sugar moieties. Monomethylated species were termed methylmycofactocinones (MMFT-n) and methylmycofactocinols (MMFT-nH 2 ), respectively. A targeted search for theoretical mass traces revealed additional members of the MFT-n(H 2 ) and MMFT-n(H 2 ) series (Data Set 2 †). As expected, the mycofactocinones exhibited MS/MS fragments with a systematic shi by À2.0016 (e.g., m/z 234.11, 396.16, 572.24) ( Fig. 2A) demonstrating that the reduction/oxidation indeed takes place in the PMFT moiety. Oligoglycosylation with up to n ¼ 9 saccharide units was detected, while seven and eight units appeared to be the most dominant forms. We observed both methylated (MMFT) and unmethylated (MFT) sugar chains, with the methylated series being more prominent. Only monomethylated species were found. Mass fragmentation of MMFTn(H 2 ) species was well in agreement with the assumption that the second sugar was the hotspot for methylation. For instance, MS/MS fragmentation of MMFT-8H 2 yielded peaks corresponding to ions of MFT-1H 2 (398.18011) and MMFT-2H 2 (574.24640) suggesting that the methyl group is present in the second sugar moiety ( Fig. 2A).

Structure elucidation of the oligosaccharide moiety
To determine the structure of elongated mycofactocins, we conducted large-scale cultivations. The dominant mycofactocin exhibited the same mass and fragmentation pattern as MMFT-2H 2 , but eluted at a slightly shied retention time (Fig. S3 †). We therefore named this species MMFT-2bH 2 . Due to the low yields and co-elution of contaminants, structural analysis by nuclear magnetic resonance (NMR) was not possible at this stage. However, cellulase (b-1,4-glucanase) treatment degraded the sugar chain of mycofactocin species (n > 2), while amylase (a-1,4-glucanase) did not exhibit any effect (Fig. 3A). This nding strongly suggested that the oligosaccharide chain represents a b-1,4-glucan. Intriguingly, isomer MMFT-2bH 2 , but not MMFT-2H 2 , accumulated aer the enzymatic digest, suggesting that MMFT-2b(H 2 ) represents the product of cellulase digestion of MMFT-7/8(H 2 ) and shares the identical disaccharide anchor.
To further elucidate the structure of MMFT-nH 2 , we analyzed enriched fractions of MMFT-2bH 2 and MMFT-nH 2 by chemical derivatization and gas chromatography coupled with mass spectrometry (GC-MS). Monosaccharides were released by acid hydrolysis and derivatized by trimethylsilylation (TMS). Comparative analysis of peaks arising from the MMFT-nH 2 and MMFT-2bH 2 fractions and carbohydrate standards conrmed the presence of D-glucose (Fig. S4 †) and revealed that the methylated sugar present in MMFT-n(H 2 ) is 2-O-methyl-Dglucose (Fig. S5 †). To conrm the glycosidic linkage positions, the oligosaccharide was permethylated before hydrolysis so that only hydroxyl groups involved in glycosidic bond formation would be free for silylation. 23 This experiment (Fig. S6 †) lead to the formation of glucose with 2,3,6-O-methyl-1,4-O-TMS modication conrming the 1,4-glycosidic linkage. Additional modication experiments (methanolysis and permethylation) supported the assignments (Fig. S7-S19 †). Aer repeated cultivation we nally obtained MMFT-7/8H 2 in sufficient amounts to record 1D and 2D-NMR spectra (ESI results and discussion, Fig. S20-S27, Tables S1 and S2 †). The 1 H NMR spectrum of MMFT-7/8H 2 exhibited a similar ve-membered lactam moiety as present in AHDP, but an isolated methine group was shied to low-eld (d H-3 4.28 ppm/d C-3 76.16 ppm) compared to AHDP (d H-3 3.30 ppm/d C-3 61.64 ppm). This indicated the amine group connected to C-3 was replaced by a hydroxyl group. The HMBC correlation between H-1 0 to C-11 suggested the sugar chain to be attached to the hydroxyl group of the tyrosine moiety (Fig. 3B). The b-1,4-glycosidic linkage was  Fig. 3C. In summary, we propose that the oligosaccharide moiety of MFT is a b-1,4-glucane (cellulose). The methylated hexose present in MMFT-n(H 2 ) and MMFT-2b(H 2 ) was shown to be 2-O-methylglucose. The fact that MMFT-2 and MMFT-2b (digested MMFT-n) are distinct in retention times points to some degree of structural diversity within MMFTs. Notably, cellulose was shown to be produced by M. tuberculosis as a constituent of biolms aer exposure to reductive stress. 24 The production of methylated glucans, like 6-O-methylglucose lipopolysaccharides (MGPL), albeit with a-1,4 linkage, is well described in Mycobacteria, 2-Omethylglucose appears to be less common. 25 Glycosylation is a relatively uncommon modication of cofactors. The most important examples are mycothiol 4 and bacillithiol. 26 Glycine-derived intermediates of MFT biosynthesis Surprisingly, the MS/MS network ( Fig. 2A) revealed two additional compounds (m/z 1426.54 and 1588.59) with an unusual mass shi compared to the MFT-n(H 2 ) candidates. Their mass differences and MS/MS spectra indicated that they represented hepta-and octaglycosylated species sharing a head moiety closely related to PMFT and PMFT(H 2 ). The molecular masses and MS/MS spectra of the compounds could be explained by the assumption that the aglycon corresponded to glycyl-AHDP (GAHDP) and these compounds represented the oligoglycosylated forms GAHDP-7 and GAHDP-8. Since the VY core peptide of MA is preceded by a glycine residue at its N-terminal side it appeared highly likely that the GAHDP-n species corresponded to premature cleavage products of the MC-processed precursor peptide. To corroborate this hypothesis, we fed M. smegmatis cultures with a combination of fully 13 C-labeled Gly-13 C 2 , Val-13 C 5 , and Tyr-13 C 9 . Indeed, GAHDPderived molecules underwent a mass shi of +15.05033 Da, indicating the incorporation of Gly-13 C 2 (+2.00671 Da) in addition to the decarboxylated Val-Tyr moiety (+13.04362 Da) (Fig. S28 †). Targeted searches for GAHDP-n as well as AHDP-n (lacking the glycyl residue) and MAHDP-n candidates (AHDP decorated with monomethylated oligosaccharide) revealed three series of oligoglycosylated compounds with similar retention times within each series (Fig. 4, Data Set 2 †).

Dissection of MFT biosynthesis
In order to test if all MFT candidate compounds were related to MFT biosynthesis, we investigated mutants (DmC, DmD, DmE, DmF) created previously 12 of the MFT biosynthesis pathway for the production of candidate molecules (Fig. 4A, Data Set 2 †). Indeed, none of the aglycons, nor any of the glycosylated candidates were detected in the DmC strain (Fig. 4B). This nding, together with the fact that the genetically complemented strain DmC-Comp restored production of MFT congeners (Data Set 2 †) represented strong evidence that we indeed identied bona-de MFT-derivatives.
The DmE mutant was able to produce mycofactocins, albeit in signicantly lower amounts, explaining the previously unexpected phenotypic observation that the DmE mutant was able to grow on ethanol, but slower than WT. 12 Intriguingly, the pool of GAHDP-n was strongly increased in the DmE strain (Fig. 4A). We thus conclude that ME can be complemented by an unknown peptidase present in the metabolic background of mycobacteria. Theoretically, an aminopeptidase would be sufficient to degrade the N-terminus of MA, releasing the AHDP-like core. Peptidases encoded outside the biosynthetic gene cluster have been observed in other RiPP biosyntheses as well. 27 However, the removal of the glycine residue might be an apparent bottleneck of the alternative maturation pathway in M. smegmatis. Alternatively, GAHDPs could represent shunt products that cannot be further processed. In full agreement with the in vitro nding that MD consumes AHDP to form PMFTH 2 , 21 all metabolites downstream of (M)AHDP-n were abrogated in the DmD strain, whereas AHDP-n and GAHDP-n accumulated (Fig. 4). The fact that GAHDP-n increased might suggest that the ME step is impeded in the absence of MD as well. Genetic dysregulation or cooperative effects between the two enzymes, like complex formation and substrate channeling, might account for this result.

Glycosylation of MFT is mediated by MF
It has been speculated that the putative glycosyltransferase MF catalyzes a nal glycosylation of PMFT (Fig. 1C) to yield the mature cofactor. 21 Our results at this point showed that multiple glucose residues are indeed attached to the aglycon in vivo. However, glycosylation appeared already at an early stage as mirrored by the presence of the glycosylated (G)AHDP-n series. In order to link oligoglycosylation to a given gene product, we analyzed the DmF mutant for the production of glycosylated MFT congeners. Indeed, all glycosylated MFT congeners were abolished in the DmF metabolome. Unexpectedly, DmF mutants additionally ceased to produce the aglycons PMFT and PMFTH 2 . MF did, however, produce trace amounts of AHDP, thus showing that at least residual MC activity was present in the mutant (Fig. 4B). To exclude polar effects, we complemented DmF by re-introduction of the mF gene under control of the mA promotor. The restoration of the full MFT metabolite spectrum (Data Set 2 †) excluded polar effects and thus veried that MF was the glycosyltransferase responsible for oligoglycosylation of MFT congeners. The appearance of glycosylated (G)AHDP species in WT together with the drastic decrease of aglycons in DmF can be interpreted in a scenario where either glycosylation or the MF protein itself are essential for the MD step to efficiently take place in vivo. If missing, the biosynthetic machinery may fail to assemble a functional complex or may be unable to recruit the unglycosylated metabolic precursors. The nding that the mF gene is a conserved constituent of MFT biosynthetic loci among different phyla supports the importance of this modication. 11 The deduced MF protein of M. smegmatis (MSMEG_1426) consists of 470 amino acids (aa) and belongs to the glycosyltransferase 2 family (GT2) according to PFAM (PF00535) and CAZy searches. These enzymes are known for an inverting mechanism of oligoglycoside formation. This is well in agreement with the proposed b-conguration of the MFT oligosaccharide chain. Sequence alignment (Fig. S29A †) showed a high degree of sequence conservation among mycobacterial species and other actinomycetes (e.g., 92% similarity to MF of M. tuberculosis H37Rv). Prediction of transmembrane domains revealed a single helix spanning residues 324-346 with the Nterminus being located outside of the membrane (Fig. S29B †). The MMFT biosynthetic machinery, however, appears not to be fully encompassed within the MFT cluster since no methyltransferase was found. Future studies are warranted to identify the enzymes involved in MFT oligosaccharide methylation.

Cofactor role of mycofactocin
Aer discovery of the glycosylated mycofactocins, we examined to which extent their production was actually dependent on the presence of ethanol in culture media. We therefore systematically compared the metabolome of M. smegmatis WT aer ethanol treatment with glucose controls (Data Set 3 †).
The results demonstrated that all MFT congeners or intermediates were strongly upregulated upon cultivation on ethanol (median: 34-fold upregulation) (Fig. 5A). These data perfectly support a recent report that MFT is involved in alcohol metabolism. 12 Finally, we sought to conrm that the MFT congeners identied in this study are indeed coenzymes of MFTdependent enzymes. To assess this question, we turned to activity-based metabolic proling. 28 We incubated the extracted metabolome of M. smegmatis with the recombinant L-carveol dehydrogenase LimC (CAB54559.1) from Rhodococcus erythropolis (Fig. S30 †), a nicotinoprotein with a nonexchangable NADH cofactor. 29 This enzyme was proposed to require MFT as an external electron acceptor. 11 A recent study showed that carveol dehydrogenase from M. smegmatis was able to reduce PMFT to PMFTH 2 using carveol and internally bound NADH as an electron donor. 21 Likewise, we observed full reduction of all mycofactocinones to mycofactocinols (Fig. 5B, Data Set 4 †) by LimC when combined with carveol as a substrate. Controls lacking enzyme or substrate showed weak and no turnover, respectively. The low turnover by LimC alone can be explained by internally bound NADH as reported before. 21 Both the aglycon PMFT as well as the oligoglycosylated MFT-n and MMFT-n species were completely turned over, while redox-inactive AHDP congeners remained unaffected. These data further validate the notion that all MFT candidates presented here are mycofactocins with full cofactor function. It remains to be claried if there is a preference for the glycosylated coenzymes or their aglycons in the bacterial cell.

Conclusion
The redox cofactor mycofactocin has attracted considerable interest since it was postulated by bioinformatics. Despite recent progress made by in vitro studies, evidence for mycofactocin congeners in living microorganisms has been missing so far. Our integrated metabolomics approach combining stable isotope labeling, metabolite induction, MS/MS networking as well as genetic dissection of the biosynthetic pathway turned out to be a powerful approach to identify RiPP congeners in bacteria and could inspire similar projects in the future. Using this technique, we discovered natural MFT and found that it is decorated with oligosaccharides consisting of up to nine b-1,4-linked glucose units. Analyses of DmF mutants and complement strains revealed that MF is the glycosyltransferase responsible for the oligoglycosylation observed. Mycofactocins can be isolated in oxidized (mycofactocinones) and reduced forms (mycofactocinols) and are co-substrates of enzymatic reduction by carveol dehydrogenase. These data provide strong evidence that mycofactocins are indeed redox cofactors as proposed earlier. 11,12,21 We, therefore, conclude that we have nally discovered the family of compounds that was tentatively called "mycofactocin" and thus close an important gap of knowledge in the eld. Our results will guide further studies into the occurrence, physiological role, and biochemistry of mycofactocins in microorganisms. Finally, these and other studies will inspire future efforts to exploit mycofactocin, e.g., as a disease marker or as a potential drug target for the treatment mycobacterial infections.

Conflicts of interest
There are no conicts to declare.