Mycofactocin Biosynthesis Proceeds through 3-Amino-5-[( p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP); Direct Observation of MftE Specificity toward MftA

Abstract

The structure of the ribosomally synthesized and post-translationally modified peptide product mycofactocin is unknown. Recently, the first step in mycofactocin biosynthesis was shown to be catalyzed by MftC in two S-adenosylmethionine-dependent steps. In the first step, MftC catalyzes the oxidative decarboxylation of the MftA peptide to produce the styrene-containing intermediate MftA**, followed by a subsequent C-C bond formation to yield the lactam-containing MftA*. Here, we demonstrate the subsequent biosynthetic step catalyzed by MftE is specific for MftA*. The hydrolysis of MftA* leads to the formation of MftA(1-28) and 3-amino-5-[( p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP). The hydrolysis reaction is Fe²⁺-dependent, and addition of the metal to the reaction mixture leads to a k_obs of ∼0.2 min^-1. Lastly, we validate the structure of AHDP by ¹H, ¹³C, and COSY nuclear magnetic resonance techniques as well as mass spectrometry.

Figure 1.

(A) A schematic representation of the mycofactocin biosynthetic pathway (blue) and the sequence of MftA used in this study. The gene encoding for a short-chain dehydrogenase (SDR) is shown in red. (B) MftC catalyzes the SAM dependent oxidative decarboxylation of the C-terminal tyrosine of MftA to form MftA** followed by a subsequent SAM dependent C-C bond formation between the penultimate valine and the β-carbon of tyrosine to form MftA*. The subsequent MftE modification is unknown and is the focus of this study.

Figure 2.

A sequence similarity network for the creatinine amidohydrolase family (IPR003785) shows the divergence of the mycofactocin peptidase (purple) and the FAPy deformylase (green) from the main body of creatinine amidohydrolases.

Figure 3.

The HPLC chromatograms of reactions containing 50 μM of MftA variant with and without 50 μM MftE, indicate that MftE preferentially hydrolyses MftA* to form two new products at retention times 12.5 min and 13.9 min (orange). All reactions were carried out in the presence of 50 μM Zn²⁺ and 50 μM Fe²⁺.

Figure 4.

(A) COSY NMR spectra of AHPD in the aromatic region showing the cross-peak coupling between hydrogens on C9-C10. (B) COSY NMR spectra of AHPD in the saturated region showing the cross-peak coupling between hydrogens on C7-C1 and C5-C1. (C) Structural representation of the results from COSY NMR. Cross-peak couplings are represented by thick bonds.