New Insights into Mycofactocin Biosynthesis, Structure and Function

Mycofactocin is a putative ribosomally synthesized and post-translationally modified peptide (RiPP)-derived redox cofactor. Its biosynthesis is accomplished through the dedicated actions of the products of six conserved genes, mftABCDEF. The mycofactocin pathway is one of the most widely distributed RiPP systems in bacteria however, this distribution is heavily skewed towards the Mycobacteria genus including human pathogenic variants such as M. tuberculosis and M. ulcerans. Gene expression studies have demonstrated the essentiality of the pathway in the ability of M. tuberculosis to utilize the host\u27s cholesterol as sole carbon source during latency. However, the biosynthesis, structure and physiological function of mycofactocin remain enigmatic. Current efforts to elucidate the biosynthesis, structure and function of mycofactocin have focused on in vitro reconstitution of each enzyme in the pathway to gain insights into their role and function. The biosynthesis of mycofactocin commences with the ribosomal production of the precursor peptide MftA containing conserved C-terminal residues - IDGMCGVY. In the presence of the RRE domain MftB, the RS-SPASM enzyme MftC, catalyzes the SAM-dependent oxidative decarboxylation and carbon-carbon bond formation on MftA to form MftA*. The roles of the auxiliary [Fe-S] clusters in MftC catalysis as well as the subsequent steps in the biosynthesis of mycofactocin are not known. Here, we have provided additional information regarding the roles of the auxiliary clusters in MftC. We showed that MftC contains three [4Fe-4S] clusters, all of which are required for catalysis. In addition, we measured the midpoint potentials of the clusters to provide insights into the redox flipping mechanism of MftC. Furthermore, we reconstituted the activity of MftE and showed that it selectively hydrolyzes MftA* to form MftA (1-28) and a 3-amino-5-(4-hydroxybenzyl)-4,4-dimethylpyrrolidin-2-one, herein referred to as AHDP. From this study, we have clarified the misunderstandings surrounding the accurate precursor for mycofactocin biosynthesis. Subsequently, we reconstituted the activity of MftD and showed that it catalyzes the oxidative deamination of AHDP to form an α-keto moiety herein referred to as premycofactocin. Lastly, we measured the midpoint potential of premycofactocin to be ~ -255 mV and demonstrated that it is used by mycofactocin-associated short chain dehydrogenases for multiple catalytic turnover

Occurrence, Function, and Biosynthesis of Mycofactocin

Mycofactocin is a member of the rapidly growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Although the mycofactocin biosynthetic pathway is widely distributed among Mycobacterial species, the structure, function, and biosynthesis of the pathway product remain unknown. This mini-review will discuss the current state of knowledge regarding the mycofactocin biosynthetic pathway. In particular, we focus on the architecture and distribution of the mycofactocin biosynthetic cluster, mftABCDEF, among the Actinobacteria phylum. We discuss the potential molecular and physiological role of mycofactocin. We review known biosynthetic steps involving MftA, MftB, MftC, and MftE and relate them to pyrroloquinoline quinone biosynthesis. Lastly, we propose the function of the remaining putative biosynthetic enzymes, MftD and MftF

Mycofactocin Biosynthesis Proceeds through 3-Amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP); Direct Observation of MftE Specificity toward 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 Fe2+-dependent, and addition of the metal to the reaction mixture leads to a kobs of ∼0.2 min–1. Lastly, we validate the structure of AHDP by 1H, 13C, and COSY nuclear magnetic resonance techniques as well as mass spectrometry

Structural Insights for Vanadium Catecholates and Iron‑sulfur Clusters Obtained from Multiple Data Analysis Methods Applied to Electron Spin Relaxation Data

Electron paramagnetic resonance (EPR) inversion recovery curves for vanadium catecholates and iron‑sulfur clusters were analyzed with three models: the sum of two exponentials, a stretched exponential, and a model-free distribution of exponentials (UPEN). For all data sets studied fits with a stretched exponential were statistically indistinguishable from the sum of two exponentials, and were significantly better than for single exponentials. UPEN provides insights into the structures of the distributions. For a vanadium(IV) tris catecholate the distribution of relaxation rates calculated with UPEN shows the contribution from spectral diffusion at low temperatures. The energy of the local mode for this complex, found from the temperature dependence of the spin lattice relaxation, is consistent with values expected for a metal-ligand vibration. For the [2Fe-2S]+ cluster in pyruvate formate lyase activating enzyme (PFL-AE) the small stretched exponential β values (0.3) at low temperature and the distributions calculated with UPEN reflect the contribution from a second rapidly relaxing species that could be difficult to detect by continuous wave EPR. The distributions in 1/T1 for the [4Fe-4S]+ clusters in Mycofactocin maturase were about a factor of four wider than for the three other systems studied. The very broad distribution of relaxation rates may be due to protein mobility and distributions in electronic energies and local environments for the clusters. UPEN provides insight into several situations that can result in low values of stretch parameter β including contributions from spectral diffusion, overlapping signals from distinguishable clusters, or very wide distributions

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C–C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe–S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe–4S] cluster and two auxiliary [4Fe–4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe–4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other

New developments in RiPP discovery, enzymology and engineering

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research