Mycofactocin biosynthesis proceeds through 3-amino-5-[(p-hydroxyphenyl) methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP); direct observation of MftE specificity towards 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(I-28) and 3-amino-5-[(p-hydroxyph enyl) 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 similar to 0.2 min(-1). Lastly, we validate the structure of AHDP by H-1, C-13, and COSY nuclear magnetic resonance techniques as well as mass spectrometry.