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.

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.