MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin

Abstract

Mycofactocin (MFT) is a putative ribosomally synthesized and post-translationally modified (RiPP) redox cofactor. The biosynthesis of MFT is encoded by the gene cluster mftABCDEF. While processing of the precursor peptide by MftB, MftC, and MftE has been shown to result in the formation of the small molecule 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP), no activity has been shown for the putative dehydrogenase MftD and the putative glycosyltransferase MftF. In addition, evidence demonstrating that MFT is a redox cofactor has only been limited to the requirement of mft genes for ethanol assimilation in Mycobacterium smegmatis mc²155. Here, we demonstrate that MftD catalyzes the oxidative deamination of AHDP, forming an α-keto moiety on the resulting molecule, which we call pre-mycofactocin (PMFT). We characterize PMFT by 1D and 2D NMR spectroscopy techniques and by high-resolution mass spectrometry data to solve its structure. We further characterized PMFT by cyclic voltammetry and found its midpoint potential to be ∼255 mV. Lastly, we demonstrate that PMFT is a biologically active redox cofactor that oxidizes NADH bound by M. smegmatis carveol dehydrogenase (MsCDH) and can be used by MsCDH in the oxidation of carveol. These data demonstrate for the first time that PMFT functions as a biologically active redox mediator and provides the most direct evidence to date that MFT is a RiPP-derived redox cofactor.

Figure 1 –

Mycofactocin biosynthetic pathway. A) An arrow representation of the mycofactocin biosynthetic pathway showing the association of a short chain dehydrogenase (SDR) and the MuMftA amino acid sequence. B) A condensed reaction scheme of known steps in mycofactocin biosynthesis. Enzymatic modifications are shown in red.

Figure 2 –

A) MftD was purified to homogeneity as determined by SDS-PAGE analysis. Lane 1 contains the Goldbio BLUEstain Protein Ladder standard, Lane 2 contains His6-purified MuMftD, and lane 3 contains His₆-purified MsMftD. B) A UV-Vis spectral analysis of anaerobically prepared MftD indicates the presence of a reduced flavin (red). Upon exposure to air of the protein sample, the UV-Vis spectrum shifts considerably, indicating that the flavin is oxidized. C) High pressure liquid chromatography analysis of the flavin containing fraction of protein precipitate (red) indicates that MftD likely binds FMN when compared to retention times of flavin standards.

Figure 3 –

A) HPLC chromatograms of reactions containing AHDP (black), AHDP and MsMftD (red), and AHDP and MuMftD (blue) indicate that AHDP is an active substrate for MftD. B) HRMS analysis of the AHDP (black) and the MftD product (blue) shows an ion with a m/z that is consistent with the loss of -NH₃ and the addition of O. C) From the HPLC and HRMS analysis, MftD is proposed to catalyze the oxidative deamination of AHDP (black) to form pre-mycofactocin (blue, PMFT). The theoretical m/z for the molecules are indicated above their structure.

Figure 4 –

NMR analysis of PMFT. A) Overlaid COSY spectra of AHDP (red) and PMFT (blue) reveals the loss of the H_α signal. B) HMBC spectra for PMFT provides evidence that the C_α has shifted downfield to ~210 ppm. C) Overlaid HSQC spectra of ADHP (red) and PMFT (blue) shows the loss of the H-C_α heteronuclear interaction. D) A reference structure of PMFT with the important carbon annotated. E) A summary of relevant NMR correlations on PMFT.

Figure 5 –

A) High resolution mass spectra of the MftD reaction carried out in ¹⁸OH₂ shows the enrichment of the ¹⁸O incorporated PMFT (red). The structure for natural abundance and enriched PMFT are shown above with their theoretical mass for the [M+H]⁺ ion. B) HPLC analysis of MftD reactions under anaerobic (blue) and aerobic (red) conditions suggest that molecular oxygen is required for catalytic turnover. The reference chromatogram for AHDP is shown in black.

Figure 6 –

A) Overlaid cyclic voltammogram of PMFT/SWCNT (red) and buffer/SWCNT (black). Voltammetry of PMFT was measured at pH 7.0 and at 22 °C with a scan rate of 50 mV/s. B) Overlaid UV-visible spectra showing the oxidation of MsCDH bound NADH by PMFT. C) HPLC analysis demonstrates that MsCDH is active towards carveol in the presence of PMFT (blue). The chromatogram for PMFT is shown in red. D) HRMS analysis of the ~14.9 min peak shows an ion with a m/z that is consistent with the mass of PMFTH₂ (theoretical [M+H]⁺ m/z = 236.1281). E) The proposed structure of PMFT following a 2e⁻/2H⁺ reduction.

Figure 7 –

Stopped-flow kinetic analysis of a single turnover oxidation reaction with 160 μM MsCDH and 25 μM (red dashes) or 50 μM (magenta dashes) PMFT. The oxidation of NADH at 340 nm was monitored during the reaction, sampling every 2 ms. Each kinetic trace is an average of 3 individual experiments. The averages were fitted to a single exponential decay (black) to determine the rate constant.

Figure 8.

The structures of the known peptide derived redox cofactors where TPQ, LTQ, TTQ, and CTQ are formed in situ of the active enzyme and PQQ and PMFT are formed from a dedicated RiPP biosynthetic pathway.

Scheme 1 –

A) A condensed reaction scheme for D-amino acid oxidase (DAAO) and B) for MftD.