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

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.