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

Abstract

Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. A preliminary biosynthetic model had been established by bioinformatics and in vitro studies, while the structure of natural MFT and key biosynthetic steps remained elusive. Here, we report the discovery of glycosylated MFT by ¹³C-labeling metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Extensive structure elucidation including NMR revealed that MFT is decorated with up to nine β-1,4-linked glucose residues including 2-O-methylglucose. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the redox cofactor function of glycosylated MFTs by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results will guide future studies into the biochemical functions and physiological roles of MFT in bacteria.

Fig. 1. Biosynthesis of mycofactocin. (A) Schematic representation of the MFT biosynthetic gene cluster of M. smegmatis. Arrows present genes mftA-F. The scale bar indicates 1000 base pairs. (B) Current biosynthesis model of MFT revealed by in vitro studies. The precursor peptide MftA (WP_029104568.1) is bound by its chaperone MftB. The rSAM enzyme MftC catalyzes oxidative decarboxylation and cyclization of the core peptide consisting of a C-terminal Val–Tyr dipeptide. The peptidase MftE releases the cyclized core to form AHDP. MftD performs oxidative deamination of AHDP yielding pre-mycofactocin (PMFT), the presumed redox-active core. (C) The putative glycosyltransferase MftF (WP_011727662.1) was hypothesized to glycosylate premycofactocins. (P)MFT is reduced to (P)MFTH₂ (mycofactocinol) by oxidoreductases. dAdo: 5′-deoxyadenosine, GMC: glucose-methanol-choline oxidoreductase, SAM: S-adenosyl methionine.

Fig. 2. Discovery and tandem mass spectrometry of MFT congeners. (A) Molecular network of MFT congeners. Nodes (circles) represent chemical compounds. Internal node labels display the precursor mass of compounds (m/z [M + H⁺]). External node labels show proposed compound annotations. Edges represent relationships in terms of shared MS/MS fragments. Edge labels show proposed modifications based on precursor mass shifts (blue: hexosylation, red: oxidation/reduction, grey: methylation). Line widths of edges mirror cosine distances. Representative MS/MS spectra of corresponding precursor ions are shown above or below nodes. (B) Schematic representation of mass fragmentation patterns of GAHDP-n, MMFT-nH₂ and MMFT-n. Numbers indicate mass-to-charge ratios (m/z) of fragments observed. Circles represent hexose moieties. Me: methyl group.

Fig. 3. Structure of mycofactocins. (A) Enzymatic degradation of MMFT-n by cellulase. Extracted ion chromatograms (XIC, [M + H]⁺) of extract of M. smegmatis corresponding to MMFT-7H₂ (m/z 1383.50626, left stack) and MMFT-2H₂ or MMFT-2bH₂ (m/z 574.24640, right stack) after treatment with cellulase, amylase, or buffer (control) are shown. Asterisk designates a peak corresponding to the M + 2 isotope of MMFT-7. Digestion by cellulase (β-1,4-glucanase) consumes MMFT-nH₂ and produces MMFT-2bH₂ suggesting that the oligosaccharide consists of β-1,4-linked glucose. (B) Key COSY and HMBC correlations of MMFT-7/8H₂. (C) Proposed chemical structures of key mycofactocins and biosynthetic congeners. Mycofactocins are glycosylated by sugar chains consisting of up to nine β-1,4-linked glucose units (n ≤ 9). In methylated mycofactocins (MMFT) the second hexose is methylated (2-O-methyl-d-glucose). The aglycon is PMFT or PMFTH₂ in mycofactocinones or mycofactocinols, respectively. The aglycon is AHDP or GAHDP in biosynthetic precursors AHDP-n and GAHDP-n, respectively.

Fig. 4. Metabolic profile of MFT congeners present in M. smegmatis. (A) Distribution of proposed MFT congeners as determined by LC-MS (Data Set 2†). Bars indicate area under the curve of designated species (average of three biological replicates, n = 3). Blue: WT, red: ΔmftE, gray: ΔmftD. The ΔmftE mutant produces significantly reduced amounts of MFT congeners compared to WT, but accumulates incorrectly cleaved products (GAHDP-n series). ΔmftD is unable to produce PMFT(H₂) and glycosylated (M)MFT-n(H₂), thus accumulating AHDP-n congeners. (B) Extracted ion chromatograms (XIC, [M + H]⁺) of WT and mutants (ΔmftC, ΔmftD, ΔmftE, ΔmftF) corresponding to AHDP (m/z 235.14411), PMFT (m/z 234.11247), PMFTH₂ (m/z 236.12812), MMFT-8 (m/z 1544.55072) and MMFT-8H₂ (m/z 1546.56637). **marks minor isomeric forms (Fig. S2†). ***marks a peak corresponding to the M + 2 isotope of MMFT-8. ΔmftC is blocked in biosynthesis of all MFT intermediates, ΔmftF abolishes most of the MFT products, but forms trace amounts of AHDP (*). ΔmftE produces most MFT species in lower amounts, while intermediates like AHDP are increasing. ΔmftD strongly accumulates AHDP, while MFT congeners are abolished.

Fig. 5. Cofactor role of mycofactocin. (A) MFT congeners are strongly upregulated (MMFT-8H₂: 26-fold) on ethanol-containing media. Area under the curve of MFT species produced by M. smegmatis treated with ethanol (dark gray) versus glucose controls (light gray) are shown (Data Set 3†). (B) Reduction of mycofactocinones to mycofactocinols by treatment of extracts with LimC and carveol. Blue: complete assay with enzyme and l-carveol as substrate. Red: control without substrate, gray: control without enzyme. Bars represent average area under the curve, error bars standard deviation of 3 biological replicates (n = 3) for in both charts.