Macrocyclization and Backbone Rearrangement During RiPP Biosynthesis by a SAM-Dependent Domain-of-Unknown-Function 692.

The domain of unknown function 692 (DUF692) is an emerging family of post-translational modification enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Members of this family are multinuclear iron-containing enzymes, and only two members have been functionally characterized to date: MbnB and TglH. Here, we used bioinformatics to select another member of the DUF692 family, ChrH, that is encoded in the genomes of the Chryseobacterium genus along with a partner protein ChrI. We structurally characterized the ChrH reaction product and show that the enzyme complex catalyzes an unprecedented chemical transformation that results in the formation of a macrocycle, an imidazolidinedione heterocycle, two thioaminals, and a thiomethyl group. Based on isotopic labeling studies, we propose a mechanism for the four-electron oxidation and methylation of the substrate peptide. This work identifies the first SAM-dependent reaction catalyzed by a DUF692 enzyme complex, further expanding the repertoire of remarkable reactions catalyzed by these enzymes. Based on the three currently characterized DUF692 family members, we suggest the family be called multinuclear non-heme iron dependent oxidative enzymes (MNIOs).

A remarkable transformation catalyzed by a domain-of-unknown-function 692 during the biosynthesis of a new RiPP natural product.

The domain of unknown function 692 (DUF692) is an emerging family of posttranslational modification enzymes involved in the biosynthesis of ribosomally-synthesized and posttranslationally modified peptide (RiPP) natural products. Members of this family are multinuclear iron-containing enzymes and only two members have been functionally characterized to date: MbnB and TglH. Here, we used bioinformatics to select another member of the DUF692 family, ChrH, that is ubiquitously encoded in the genomes of the Chryseobacterium genus along with a partner protein ChrI. We structurally characterized the ChrH reaction product and show that the enzyme catalyzes an unprecedented chemical transformation that results in the formation of a macrocycle, an imidazolidinedione heterocycle, two thioaminals, and a thiomethylation. Based on isotopic labeling studies, we propose a mechanism for the four-electron oxidation and methylation of the substrate peptide. This work identifies the first SAM-dependent DUF692 enzyme, further expanding the repertoire of remarkable reactions catalyzed by these enzymes.

A scalable platform to discover antimicrobials of ribosomal origin.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.

Peptide backbone modifications in lanthipeptides.

Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products characterized by the presence of lanthionine and methyllanthionine. During the maturation of select lanthipeptides, five different alterations have been observed to the chemical structure of the peptide backbone. First, dehydratases generate dehydroalanine and dehydrobutyrine from Ser or Thr residues, respectively. A second example of introduction of unsaturation is the oxidative decarboxylation of C-terminal Cys residues catalyzed by the decarboxylase LanD. Both modifications result in loss of chirality at the α-carbon of the amino acid residues. Attack of a cysteine thiol onto a dehydrated amino acid results in thioether crosslink formation with either inversion or retention of the l-stereochemical configuration at the α-carbon of former Ser and Thr residues. A fourth modification of the protein backbone is the hydrogenation of dehydroamino acids to afford d-amino acids catalyzed by NAD(P)H-dependent reductases. A fifth modification is the conversion of Asp to isoAsp. Herein, the methods used to produce and characterize the lanthipeptide bicereucin will be described in detail along with a brief overview of other lanthipeptides.

Structural insights for vanadium catecholates and iron­sulfur clusters obtained from multiple data analysis methods applied to electron spin relaxation data.

Electron paramagnetic resonance (EPR) inversion recovery curves for vanadium catecholates and iron­sulfur 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 ß 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 ß including contributions from spectral diffusion, overlapping signals from distinguishable clusters, or very wide distributions.

MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin.

Mycofactocin (MFT) is a putative ribosomally synthesized and post-translationally modified (RiPP) redox cofactor. The biosynthesis of MFT is encoded by the gene cluster mftABCDEF. While processing of the precursor peptide by MftB, MftC, and MftE has been shown to result in the formation of the small molecule 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP), no activity has been shown for the putative dehydrogenase MftD and the putative glycosyltransferase MftF. In addition, evidence demonstrating that MFT is a redox cofactor has only been limited to the requirement of mft genes for ethanol assimilation in Mycobacterium smegmatis mc2155. Here, we demonstrate that MftD catalyzes the oxidative deamination of AHDP, forming an α-keto moiety on the resulting molecule, which we call pre-mycofactocin (PMFT). We characterize PMFT by 1D and 2D NMR spectroscopy techniques and by high-resolution mass spectrometry data to solve its structure. We further characterized PMFT by cyclic voltammetry and found its midpoint potential to be ∼255 mV. Lastly, we demonstrate that PMFT is a biologically active redox cofactor that oxidizes NADH bound by M. smegmatis carveol dehydrogenase (MsCDH) and can be used by MsCDH in the oxidation of carveol. These data demonstrate for the first time that PMFT functions as a biologically active redox mediator and provides the most direct evidence to date that MFT is a RiPP-derived redox cofactor.

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.

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.

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.

Mechanistic elucidation of the mycofactocin-biosynthetic radical S-adenosylmethionine protein, MftC.

Ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways produce a diverse array of natural products. A subset of these pathways depends on radical S-adenosylmethionine proteins to modify the RiPP-produced peptide. Mycofactocin biosynthesis is one example of an S-adenosylmethionine protein-dependent RiPP pathway. Recently, it has been shown that MftC catalyzes the oxidative decarboxylation of the C-terminal tyrosine (Tyr-30) on the mycofactocin precursor peptide MftA; however, this product has not been verified by techniques other than MS. Herein, we provide a more detailed study of MftC catalysis and report a revised mechanism for MftC chemistry. We show that MftC catalyzes the formation of two isomeric products. Using a combination of MS, isotope labeling, and 1H and 13C NMR techniques, we established that the major product, MftA*, is a tyramine-valine-cross-linked peptide formed by MftC through two S-adenosylmethionine-dependent turnovers. In addition, we show that the hydroxyl group on MftA Tyr-30 is required for MftC catalysis. Furthermore, we show that a substitution in the penultimate MftA Val-29 position causes the accumulation of an MftA** minor product. The 1H NMR spectrum indicates that this minor product contains an αß-unsaturated bond that likely arises from an aborted intermediate of MftA* synthesis. The finding that MftA* is the major product formed during MftC catalysis could have implications for the further elucidation of mycofactocin biosynthesis.