FIG 2.

Mutagenic dissection of the F₄₂₀ biosynthesis pathway in M. smegmatis reveals that DH-F₄₂₀-0 is the biosynthetic intermediate in mycobacteria. (A) A schematic of the F₄₂₀ biosynthesis pathway in M. smegmatis with PEP, rather than 2PL, utilized by FbiD to create the reaction intermediate EPPG. The enzymes responsible for catalytic steps are shown, along with the 2D structures of proposed pathway intermediates and mature F₄₂₀. The yellow box highlights the reduction of DH-F₄₂₀-0, proposed to be mediated by the C-terminal domain of FbiB using FMNH₂. (B to D) LC-MS detection of mature F₄₂₀ species (B), Fo (C), and DH-F₄₂₀-0 (D) in M. smegmatis cell lysates of the wild type (Wt) and F₄₂₀ biosynthesis pathway mutants confirming the proposed function of the F₄₂₀ biosynthetic genes detecting the novel intermediate DH-F₄₂₀-0 in whole cells. F₄₂₀-X species in panel B correspond to different lengths of the polyglutamate chain where X = n tail length.