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(1–28) and 3-amino-5-[(p-hydroxyphenyl) methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP). The hydrolysis reaction is Fe 2+ dependent and addition of the metal to the reaction leads to a k obs ~ 0.2 min −1 . Lastly, we validate the structure of AHDP by a 1 H, 13 C, and COSY NMR techniques as well as mass spectrometry.