Biosynthesis of the redox cofactor mycofactocin comprises oligoglycosylation by MftF in Mycolicibacterium smegmatis

Abstract

Mycofactocin (MFT) is a redox cofactor involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. In recent years, a preliminary biosynthetic model of MFT has been established by in-vitro studies, while the final structure of MFT remained elusive. Here, we report the discovery of MFT by metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Structure elucidation revealed that MFT is decorated with up to nine b-1,4-linked glucose residues. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the cofactor function of MFT by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results close an important gap of knowledge, will guide future studies into the physiological roles of MFT in bacteria and may inspire its utilization as a biomarker or potential drug target to combat mycobacterial diseases.Keywords: Mycofactocin / cofactor / Mycobacterium / metabolomics / mass spectrometry (MS) / alcohol metabolism / drug target

New developments in RiPP discovery, enzymology and engineering

“…Indeed, its redox properties are not unlike other known peptide-derived redox cofactors, 21 and several studies have supported a redox cofactor role for mycofactocin. [266][267][268] At present it is not known how the reduced pre-mycofactocin might be reoxidized for another turnover, but recent metabolomics studies detected both oxidized and reduced glycosylated mycofactocin derivatives in Mycolicibacterium smegmatis. 266 Mycofactocin represents another class of RiPP initially discovered bioinformatically (other examples are in Sections 2.12-2.15, 3.14, and 3.24).…”