Mycofactocin biosynthesis: modification of the peptide MftA by the radical S‐adenosylmethionine protein MftC

Abstract

Mycofactocin is a putative, peptide derived, cofactor that is associated primarily with the Mycobacterium genera including the pathogen M. tuberculosis. The pathway consists of the three genes mftA, mftB, and mftC that encode for the peptide substrate, peptide chaperone, and a radical S-adenosylmethionine protein (RS), respectively. Here, we show that the MftB acts as a peptide chaperone, binding MftA with a submicromolar K D (~100 nM) and MftC with a low micromolar K D (~2 lM). Moreover, we demonstrate that MftC is a radical S-adenosylmethionine (SAM) enzyme. Finally, we show that MftC catalyzes the oxidative decarboxylation of the peptide MftA.

Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

“…These include an open reading frame encoding the small peptide precursor of mycofactocin (MftA, TIGR03969), a carrier protein (MftB, TIGR03967), a radical SAM protein (MftC, TIGR03962), a glycosyltransferase (TIGR03965), and one or more redox proteins. Interactions of MftA, MftB, MftC as well as the decarboxylation of MftA by the MftC/MftB complex have been reported recently67. In addition, most of these gene clusters also include a short chain dehydrogenase/reductase (SDR) from TIGR03971, the protein family focus of the current research.…”

“…Members of TIGR03971 appear in gene clusters near mycofactocin system protein genes mftA, mftB, and mftC567. For example, in M. paratuberculosis a putative carveol dehydrogenase (MAP_RS21280, UniProt ID Q73SC8, SSGCID target ID MypaA.01326.b) is encoded near genes for the mycofactocin precursor MftA (MAP_RS21310), the mycofactocin modification chaperone MftB (MAP_RS21315), the mycofactocin radical SAM maturase MftC (MAP_RS21320), the mycofactocin system heme/Flavin oxidoreductase MftD (MAP_RS21325), and the mycofactocin system creatininase family protein (MAP_RS21330).…”

“…Recently, the recombinant expression and characterization of MftA, MftB, and MftC have been reported67. The chaperone MftB was reported to bind the mycofactocin precursor MftA with an affinity of approximately 120 nM and the rSAM MftC with an affinity of approximately 2 μM.…”

At the confluence of ribosomally synthesized peptide modification and radical S-adenosylmethionine (SAM) enzymology

“…In the mycofactocin biosynthetic pathway, the peptide chaperone is a stand-alone protein (MftB), and in the thurincin H biosynthetic pathway, the peptide chaperone is fused to the N terminus of the RS protein (ThnB). Although morphologically different, it was shown in both systems that the peptide chaperone protein/domain is required for catalytic turnover by the RS protein (28,29,35). These findings led us to the hypothesis that the PqqD domain is ubiquitous among the remaining characterized RS-SPASM proteins (excluding AnSME).…”

“…Mycofactocin is predicted to be a redox cofactor used by a niche set of dehydrogenases found largely in the Mycobacterium genera (26,27). Initially, it was thought that MftC catalyzed the oxidative decarboxylation of the C-terminal tyrosine found on the peptide MftA, resulting in an ␣␤-unsaturated bond (28,29). However, a more detailed mechanistic study demonstrated that the decarboxylated peptide is only an intermediate of a two-step reaction (30).…”