Mechanistic elucidation of the mycofactocin-biosynthetic radical S-adenosylmethionine protein, MftC

Abstract

Ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways produce a diverse array of natural products. A subset of these pathways depends on radical S-adenosylmethionine proteins to modify the RiPP-produced peptide. Mycofactocin biosynthesis is one example of an S-adenosylmethionine protein-dependent RiPP pathway. Recently, it has been shown that MftC catalyzes the oxidative decarboxylation of the C-terminal tyrosine (Tyr-30) on the mycofactocin precursor peptide MftA; however, this product has not been verified by techniques other than MS. Herein, we provide a more detailed study of MftC catalysis and report a revised mechanism for MftC chemistry. We show that MftC catalyzes the formation of two isomeric products. Using a combination of MS, isotope labeling, and ¹H and ¹³C NMR techniques, we established that the major product, MftA*, is a tyramine-valine-cross-linked peptide formed by MftC through two S-adenosylmethionine-dependent turnovers. In addition, we show that the hydroxyl group on MftA Tyr-30 is required for MftC catalysis. Furthermore, we show that a substitution in the penultimate MftA Val-29 position causes the accumulation of an MftA** minor product. The ¹H NMR spectrum indicates that this minor product contains an αβ-unsaturated bond that likely arises from an aborted intermediate of MftA* synthesis. The finding that MftA* is the major product formed during MftC catalysis could have implications for the further elucidation of mycofactocin biosynthesis.

Keywords: MftC; S-adenosylmethionine (SAM); enzyme mechanism; iron-sulfur protein; mycofactocin; peptide biosynthesis; radical.

Figure 1.

Previously proposed reaction mechanism for MftC.

Figure 2.

MftC catalyzed the formation of two MftA products annotated as MftA* and MftA**. A, HPLC analysis of the reactions shows the conversion of MftA M1W (black) to MftA* (14.3 min) and MftA** (14.5 min). Analyses were carried out on a C4 HPLC column. B, the absorbance spectra of MftA M1W (black) and MftA* (red) are similar, whereas the absorbance spectra for MftA** (blue) indicates the appearance of a new spectral feature from 300–325 nm and a red-shift in peak maximum. mAu, milliabsorbance units. *, MftA*; **, MftA**.

Figure 3.

¹³C (A) and ¹H (B) NMR analyses of MftA* did not indicate that an αβ-unsaturated bond is present. MftA is represented by black spectra, and MftA* is represented by red spectra.

Figure 4.

Analysis of the mass spectrum of Val-29 (D₈)-labeled MftA M1W-purified peptide (A) and Val-29 (D₈)-labeled MftA M1W* (B) indicated that Val-29 forms a new intramolecular bond. Mass spectra of Val-29 (3-D)-labeled MftA M1W (C) and Val-29 (3-D) labeled MftA M1W* (D) indicates that the new intramolecular bond is formed on the C_β of Val-29. All collected spectral data are represented in black, and the predicted spectra for the deuterium-labeled MftA MH³⁺ ions are shown in red. The predicted spectra for unlabeled MftA MH³⁺ is shown in blue for panels A and B.

Figure 5.

The MftA M1W/V29A mutant led to the accumulation of MftA M1W/V29A** product. HPLC analysis of the reactions shows the conversion of MftA M1W/V29A (black) to MftA* (13.7 min) and MftA** (14.1 min). Analyses were carried out on a C4 HPLC column. B, the absorbance spectra of MftA M1W/V29A (black), MftA M1W/V29A* (red), and MftA M1W/V29A** (blue) were similar to what was found for reactions with MftA M1W. C, ¹H NMR analysis of MftA M1W/V29A** demonstrated the formation of an αβ-unsaturated indicated by appearance of doublets at ∼6.3 and ∼7.2 ppm (indicated by **). mAu, milliabsorbance units.

Figure 6.

Shown are HRMS analyses of dAdo control (A), simulated incorporation of one deuterium in dAdo (B), dAdo isolated from a reaction with Tyr-30 (ring-2,6-D₂, 2-D) MftA (C_α-labeled) (C), dAdo isolated from a reaction with Tyr-30 (3,3-D₂) MftA (C_β-labeled) (D), and dAdo isolated from a reaction with Val-29 (3-D) MftA (C_β-labeled) (E).

Figure 7.

MftC catalyzed the conversion of MftA** to MftA*. HPLC analysis of MftA** starting material (blue) and of a reaction with MftC (red) indicates a change in retention time and absorbance spectrum (inset). Analyses were carried out on a C4 HPLC column. mAu, milliabsorbance units.

Figure 8.

HPLC, UV-visible, and HRMS analysis of MftA M1W (A), MftA M1W/Y30F (B), MftA M1W/Y30S (C), and MftA M1W/Y30W (D). Each variant has a corresponding HPLC chromatogram (middle column), a UV-visible spectrum (middle column, inset), and an ion chromatogram (right column). HPLC analyses were carried out on a C4 HPLC column. For all HPLC and HRMS chromatograms, black is the unreacted MftA variant, and red represents the reaction with the MftA variant. For the UV-visible spectra, black is the unreacted MftA variant, red is the reacted MftA variant, and cyan is dAdo adduct to the Y30W variant. mAu, milliabsorbance units.

Figure 9.

Revised mechanism for MftC catalysis.