Abstract
Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. A preliminary biosynthetic model had been established by bioinformatics and in vitro studies, while the structure of natural MFT and key biosynthetic steps remained elusive. Here, we report the discovery of glycosylated MFT by 13C-labeling metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Extensive structure elucidation including NMR revealed that MFT is decorated with up to nine β-1,4-linked glucose residues including 2-O-methylglucose. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the redox cofactor function of glycosylated MFTs by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results will guide future studies into the biochemical functions and physiological roles of MFT in bacteria.
Highlights
Coenzymes are small molecules indispensable for the catalytic activity of many enzymes
Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis
While coenzymes like NAD+ or FAD are ubiquitous in nature and are essential for the core metabolism of all forms of life, specialized cofactors like pyrroloquinoline quinone (PQQ)[1] and coenzyme F420 2 are restricted to certain microbial phyla, but typically involved in extraordinary metabolic processes like methylotrophy, methanogenesis, or detoxi cation processes
Summary
Coenzymes are small molecules indispensable for the catalytic activity of many enzymes. The architecture of the MFT gene cluster (Fig. 1A) suggested that the resulting natural product is a ribosomally synthesized and post-translationally modi ed peptide (RiPP).[14] Several in vitro studies have contributed to a preliminary biosynthetic model of MFT (Fig. 1B): the precursor peptide M A of M. smegmatis consisting of 31 amino acids is produced by the ribosome and bound by its chaperone M B.
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