Small-molecule ion channels increase host defences in cystic fibrosis airway epithelia.

Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) compromise epithelial HCO3- and Cl- secretion, reduce airway surface liquid pH, and impair respiratory host defences in people with cystic fibrosis1-3. Here we report that apical addition of amphotericin B, a small molecule that forms unselective ion channels, restored HCO3- secretion and increased airway surface liquid pH in cultured airway epithelia from people with cystic fibrosis. These effects required the basolateral Na+, K+-ATPase, indicating that apical amphotericin B channels functionally interfaced with this driver of anion secretion. Amphotericin B also restored airway surface liquid pH, viscosity, and antibacterial activity in primary cultures of airway epithelia from people with cystic fibrosis caused by different mutations, including ones that do not yield CFTR, and increased airway surface liquid pH in CFTR-null pigs in vivo. Thus, unselective small-molecule ion channels can restore host defences in cystic fibrosis airway epithelia via a mechanism that is independent of CFTR and is therefore independent of genotype.

Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins.

SignificanceThe channel-forming proteusins are bacterial helical peptides that allow permeation of positively charged ions to influence membrane potential and cellular physiology. We biochemically characterize the effect of two critical posttranslational modifications on the secondary structure of the peptide substrate. We determine how a methyl group can be added to the side chains of D-Asn residues in a peptide substrate and show how flanking residues influence selectivity. These studies should foster the development of small-molecule peptide ion channels as therapeutics.

Tryptophan-Centric Bioinformatics Identifies New Lasso Peptide Modifications.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by a macrolactam linkage between the N-terminus and the side chain of an internal aspartic acid or glutamic acid residue. Instead of adopting a branched-cyclic conformation, lasso peptides are "threaded", with the C-terminal tail passing through the macrocycle to present a kinetically trapped rotaxane conformation. The availability of enhanced bioinformatics methods has led to a significant increase in the number of secondary modifications found on lasso peptides. To uncover new ancillary modifications in a targeted manner, a bioinformatic strategy was developed to discover lasso peptides with modifications to tryptophan. This effort identified numerous putative lasso peptide biosynthetic gene clusters with core regions of the precursor peptides enriched in tryptophan. Parsing of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered several putative ancillary modifying enzymes, including halogenases and dimethylallyltransferases expected to act upon Trp. Characterization of two gene products yielded a lasso peptide with two 5-Cl-Trp modifications (chlorolassin) and another bearing 5-dimethylallyl-Trp and 2,3-didehydro-Tyr modifications (wygwalassin). Bioinformatic analysis of the requisite halogenase and dimethylallyltransferase revealed numerous other putative Trp-modified lasso peptides that remain uncharacterized. We anticipate that the Trp-centric strategy reported herein may be useful in discovering ancillary modifications for other RiPP classes and, more generally, guide the functional prediction of enzymes that act on specific amino acids.

Unexpected Transformations during Pyrroloiminoquinone Biosynthesis.

Pyrroloiminoquinone-containing natural products have long been known for their biological activities. They are derived from tryptophan, but their biosynthetic pathways have remained elusive. Studies on the biosynthetic gene cluster (BGC) that produces the ammosamides revealed that the first step is attachment of Trp to the C-terminus of a scaffold peptide in an ATP- and tRNA-dependent manner catalyzed by a PEptide Aminoacyl-tRNA Ligase (PEARL). The indole of Trp is then oxidized to a hydroxyquinone. We previously proposed a chemically plausible and streamlined pathway for converting this intermediate to the ammosamides using additional enzymes encoded in the BGC. In this study, we report the activity of four additional enzymes from two gene clusters, which show that the previously proposed pathway is incorrect and that Nature's route toward pyrroloiminoquinones is much more complicated. We demonstrate that, surprisingly, amino groups in pyrroloiminoquinones are derived from (at least) three different sources, glycine, asparagine, and leucine, all introduced in a tRNA-dependent manner. We also show that an FAD-dependent putative glycine oxidase (Amm14) is required for the process that incorporates the nitrogens from glycine and leucine and that a quinone reductase is required for the incorporation of asparagine. Additionally, we provide the first insights into the evolutionary origin of the PEARLs as well as related enzymes, such as the glutamyl-tRNA-dependent dehydratases involved in the biosynthesis of lanthipeptides and thiopeptides. These enzymes appear to all have descended from the ATP-GRASP protein family.

Complete Biochemical Characterization of Pantaphos Biosynthesis Highlights an Unusual Role for a SAM-Dependent Methyltransferase.

Pantaphos is small molecule virulence factor made by the plant pathogen Pantoea ananatis. An 11 gene operon, designated hvr for high virulence, is required for production of this phosphonic acid natural product, but the metabolic steps used in its production have yet to be established. Herein, we determine the complete biosynthetic pathway using a combination of bioinformatics, in vitro biochemistry and in vivo heterologous expression. Only 6 of the 11 hvr genes are needed to produce pantaphos, while a seventh is likely to be required for export. Surprisingly, the pathway involves a series of O-methylated intermediates, which are then hydrolyzed to produce the final product. The methylated intermediates are produced by an irreversible S-adenosylmethione (SAM)-dependent methyltransferase that is required to drive a thermodynamically unfavorable dehydration in the preceding step, a function not previously attributed to members of this enzyme class. Methylation of pantaphos by the same enzyme is also likely to limit its toxicity in the producing organism. The pathway also involves a novel flavin-dependent monooxygenase that differs from homologous proteins due to its endogenous flavin-reductase activity. Heterologous production of pantaphos by Escherichia coli strains expressing the minimal gene set strongly supports the in vitro biochemical data.

A Novel Pathway for Biosynthesis of the Herbicidal Phosphonate Natural Product Phosphonothrixin Is Widespread in Actinobacteria.

Phosphonothrixin is an herbicidal phosphonate natural product with an unusual, branched carbon skeleton. Bioinformatic analyses of the ftx gene cluster, which is responsible for synthesis of the compound, suggest that early steps of the biosynthetic pathway, up to production of the intermediate 2,3-dihydroxypropylphosphonic acid (DHPPA) are identical to those of the unrelated phosphonate natural product valinophos. This conclusion was strongly supported by the observation of biosynthetic intermediates from the shared pathway in spent media from two phosphonothrixin producing strains. Biochemical characterization of ftx-encoded proteins confirmed these early steps, as well as subsequent steps involving the oxidation of DHPPA to 3-hydroxy-2-oxopropylphosphonate and its conversion to phosphonothrixin by the combined action of an unusual heterodimeric, thiamine-pyrophosphate (TPP)-dependent ketotransferase and a TPP-dependent acetolactate synthase. The frequent observation of ftx-like gene clusters within actinobacteria suggests that production of compounds related to phosphonothrixin is common within these bacteria. IMPORTANCE Phosphonic acid natural products, such as phosphonothrixin, have great potential for biomedical and agricultural applications; however, discovery and development of these compounds requires detailed knowledge of the metabolism involved in their biosynthesis. The studies reported here reveal the biochemical pathway phosphonothrixin production, which enhances our ability to design strains that overproduce this potentially useful herbicide. This knowledge also improves our ability to predict the products of related biosynthetic gene clusters and the functions of homologous enzymes.

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element.

The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded ß-sheet to form a hydrophobic ß-sandwich-like interface. The bioinformatics-guided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain-domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

Three-Center M-H-B Bonds Are Strong Field Interactions. Synthesis and Characterization of M(CH2NMe2BH3)3 Complexes of Titanium, Chromium, and Cobalt.

We describe new compounds of stoichiometry M(CH2NMe2BH3)3 (M = Ti, Cr, and Co), each of which contains three chelating boranatodimethylaminomethyl (BDAM) ligands. In all three compounds, the BDAM anion, which is isoelectronic and isostructural with the neopentyl group, is bound to the metal center at one end by a metal-carbon σ bond and at the other by one three-center M-H-B interaction. The crystal structures show that the d1 titanium(III) compound is trigonal prismatic (or eight-coordinate, if two longer-ranged M···H interactions with the BH3 groups are included), whereas the d3 chromium(III) compound and the d6 cobalt(III) compounds are both fac-octahedral. The Cr and Co compounds exhibit two rapid dynamic processes in solution: exchange between the Δ and Λ enantiomers and exchange of the terminal and bridging hydrogen atoms on boron. For the Co complex, the barrier for Δ/Λ exchange (ΔG⧧298 = 10.1 kcal mol-1) is significantly smaller than those seen in other octahedral cobalt(III) compounds; DFT calculations suggest that Bailar twist and dissociative pathways for Δ/Λ exchange are both possible mechanisms. The UV-vis absorption spectra of the cobalt(III) and chromium(III) species show that the ligand field splittings Δo caused by the M-H-B interactions are unexpectedly large, thus placing them high on the spectrochemical series (near ammonia and alkyl groups); their nephelauxetic effect is also large. The DFT calculations suggest that these properties of M-H-B interactions are in part a consequence of their three-center nature, which delocalizes electron density away from the metal center and reduces electron-electron repulsions.

Spectroscopic Investigation of Phosphorus Mineralization as Affected by the Calcite-Water Interfacial Chemistry.

The mineralization and bioavailability of phytic acid, the predominant organic phosphorus (OP) species in many soils, have generally been rendered limited due to its interaction with soil minerals. In particularly calcareous and neutral to slightly alkaline soils, phytic acid is known to actively react with calcite, although how this interaction affects phytic acid mineralization is still unknown. This study, therefore, investigated the mechanisms regarding how the calcite-water interface influences phytic acid mineralization by phytase, at pHs 6 and 8 using in situ spectroscopic techniques including solution nuclear magnetic resonance and attenuated total reflection Fourier transform infrared spectroscopy. The findings indicated a pH-specific effect of the calcite-water interface. Inhibited phytase activity and thus impaired phytic acid mineralization were induced by calcite at pH 6, while the opposite effect was observed at pH 8. How the interaction between phytic acid and calcite and between phytase and calcite differed between the two pH values contributed to the pH-specific effect. The results demonstrate the importance of soil pH, enzyme-, and OP-clay mineral interactions in controlling the mineralization and transformation of OP and, consequently, the release of phosphate in soils. The findings can also provide implications for the management of calcite-rich and limed soils.

Mechanically Triggered Carbon Monoxide Release with Turn-On Aggregation-Induced Emission.

Polymers that release functional small molecules under mechanical stress potentially serve as next-generation materials for catalysis, sensing, and mechanochemical dynamic therapy. To further expand the function of mechanoresponsive materials, the discovery of chemistries capable of small molecule release are highly desirable. In this report, we detail a nonscissile bifunctional mechanophore (i.e., dual mechano-activated properties) based on a unique mechanochemical reaction involving norborn-2-en-7-one (NEO). One property is the release of carbon monoxide (CO) upon pulsed solution ultrasonication. A release efficiency of 58% is observed at high molecular weights (Mn = 158.8 kDa), equating to ∼154 molecules of CO released per chain. The second property is the bright cyan emission from the macromolecular product in its aggregated state, resulting in a turn-on fluorescence readout coincident with CO release. This report not only demonstrates a unique strategy for the release of small molecules in a nonscissile way but also guides future designs of force-responsive aggregation-induced emission (AIE) luminogens.

Class V Lanthipeptide Cyclase Directs the Biosynthesis of a Stapled Peptide Natural Product.

Lanthipeptides are a class of cyclic peptides characterized by the presence of one or more lanthionine (Lan) or methyllanthionine (MeLan) thioether rings. These cross-links are produced by α,ß-unsaturation of Ser or Thr residues in peptide substrates by dehydration, followed by a Michael-type conjugate addition of Cys residues onto the dehydroamino acids. Lanthipeptides may be broadly classified into at least five different classes, and the biosynthesis of classes I-IV lanthipeptides requires catalysis by LanC cyclases that control both the site-specificity and the stereochemistry of the conjugate addition. In contrast, there are no current examples of LanCs that occur in class V biosynthetic clusters, despite the presence of lanthionine rings in these compounds. In this work, bioinformatics-guided co-occurrence analysis identifies more than 240 putative class V lanthipeptide clusters that contain a LanC cyclase. Reconstitution studies demonstrate that the cyclase-catalyzed product is notably distinct from the product formed spontaneously. Stereochemical analysis shows that the cyclase diverts the final product to a configuration that is distinct from one that is energetically favored. Structural characterization of the final product by multi-dimensional NMR spectroscopy reveals that it forms a helical stapled peptide. Mutational analysis identified a plausible order for cyclization and suggests that enzymatic rerouting to the final structure is largely directed by the construction of the first lanthionine ring. These studies show that lanthipeptide cyclases are needed for the biosynthesis of some constrained peptides, the formations of which would otherwise be energetically unfavored.

13C NMR Signal Enhancement Using Parahydrogen-Induced Polarization Mediated by a Cobalt Hydrogenation Catalyst.

The use of a cobalt-based catalyst for the generation of hyperpolarized 13C NMR resonances by parahydrogenation of ethyl acrylate is presented herein. Comparisons of the carboxylate 13C NMR signal enhancement factor of ethyl propionate between using (MesCCC)Co-py and a commonly utilized cationic diphosphine rhodium complex demonstrates that the cobalt system is a viable PHIP catalyst alternative. Furthermore, the operative hydrogenation mechanism of the cobalt system was examined by using 1H, 13C, and parahydrogen-induced polarization NMR spectroscopies to elucidate reaction intermediates affiliated with the observed 1H and 13C NMR signal enhancements in ethyl propionate.

Expired Epinephrine Maintains Chemical Concentration and Sterility.

OBJECTIVES: Epinephrine shortages affect nearly all American emergency medical services (EMS) systems. Utilization of expired epinephrine could mitigate this situation in daily EMS operations. Concerns about using expired medications include sterility, potency, and potential harmful chemical decay byproducts. There are no cross-platform analyses of sterility and chemical purity of multiple samples of expired parenteral epinephrine. We hypothesized that epinephrine injections will remain sterile and will retain their active ingredient's content for more than 30 months past expiration. METHODS: Six parenteral epinephrine prefilled syringes, 1 mg/10 mL, with an expiration date of January 1, 2012 had been stored in the climate controlled setting of a hospital inpatient pharmacy where they remained until they were taken for chemical or microbial analysis 30 months after expiration. An unexpired parenteral epinephrine prefilled syringe content was used as a control. Contents of three separate syringes with expired content from the same lot and one control underwent ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) and nuclear magnetic resonance (NMR) to determine epinephrine content and stability. In parallel, contents of another three expired epinephrine syringes were analyzed for sterility by plating on aerobic, anaerobic, and fungal media in a hospital microbiology laboratory. The aerobic plates were checked for growth in 3 days, the anaerobic in 5 days, and the fungal in 28 days. RESULTS: UHPLC-MS and NMR showed that content of epinephrine present in the original sample remained unchanged compared to the control. There was no statistical difference in the UHPLC-MS and NMR signal amplitudes between the control and the expired samples. No chemical degradation byproducts were detected using NMR. There was no growth of any bacteria or fungus. CONCLUSION: Recurrent epinephrine shortages impact EMS and hospital operations in the United States. Individual administrators may be hesitant to authorize use of expired pharmaceuticals due to perceived potential complications or fear of litigation. This study shows that the original parenteral epinephrine remains sterile and detectably pure more than 2.5 years after expiration. Further study of the sterility and chemical integrity of expired medications that had been subjected to the conditions of EMS vehicles may be a future research endeavor based on the aforementioned paradigm.

Well-Defined Cobalt(I) Dihydrogen Catalyst: Experimental Evidence for a Co(I)/Co(III) Redox Process in Olefin Hydrogenation.

The synthesis of a cobalt dihydrogen Co(I)-(H2) complex prepared from a Co(I)-(N2) precursor supported by a monoanionic pincer bis(carbene) ligand, (Mes)CCC ((Mes)CCC = bis(mesityl-benzimidazol-2-ylidene)phenyl), is described. This species is capable of H2/D2 scrambling and hydrogenating alkenes at room temperature. Stoichiometric addition of HCl to the Co(I)-(N2) cleanly affords the Co(III) hydridochloride complex, which, upon the addition of Cp2ZrHCl, evolves hydrogen gas and regenerates the Co(I)-(N2) complex. Furthermore, the catalytic olefin hydrogenation activity of the Co(I) species was studied by using multinuclear and parahydrogen (p-H2) induced polarization (PHIP) transfer NMR studies to elucidate catalytically relevant intermediates, as well as to establish the role of the Co(I)-(H2) in the Co(I)/Co(III) redox cycle.

The Interplay of Al and Mg Speciation in Advanced Mg Battery Electrolyte Solutions.

Mg batteries are an attractive alternative to Li-based energy storage due to the possibility of higher volumetric capacities with the added advantage of using sustainable materials. A promising emerging electrolyte for Mg batteries is the magnesium aluminum chloride complex (MACC) which shows high Mg electrodeposition and stripping efficiencies and relatively high anodic stabilities. As prepared, MACC is inactive with respect to Mg deposition; however, efficient Mg electrodeposition can be achieved following an electrolytic conditioning process. Through the use of Raman spectroscopy, surface enhanced Raman spectroscopy, (27)Al and (35)Cl nuclear magnetic resonance spectroscopy, and pair distribution function analysis, we explore the active vs inactive complexes in the MACC electrolyte and demonstrate the codependence of Al and Mg speciation. These techniques report on significant changes occurring in the bulk speciation of the conditioned electrolyte relative to the as-prepared solution. Analysis shows that the active Mg complex in conditioned MACC is very likely the [Mg2(µ-Cl)3·6THF](+) complex that is observed in the solid state structure. Additionally, conditioning creates free Cl(-) in the electrolyte solution, and we suggest the free Cl(-) adsorbs at the electrode surface to enhance Mg electrodeposition.

New reactions of terminal hydrides on a diiron dithiolate.

Mechanisms for biological and bioinspired dihydrogen activation and production often invoke the intermediacy of diiron dithiolato dihydrides. The first example of such a Fe2(SR)2H2 species is provided by the complex [(term-H)(µ-H)Fe2(pdt)(CO)(dppv)2] ([H1H](0)). Spectroscopic and computational studies indicate that [H1H](0) contains both a bridging hydride and a terminal hydride, which, notably, occupies a basal site. The synthesis begins with [(µ-H)Fe2(pdt)(CO)2(dppv)2](+) ([H1(CO)](+)), which undergoes substitution to afford [(µ-H)Fe2(pdt)(CO)(NCMe)(dppv)2](+) ([H1(NCMe)](+)). Upon treatment of [H1(NCMe)](+) with borohydride salts, the MeCN ligand is displaced to afford [H1H](0). DNMR (EXSY, SST) experiments on this complex show that the terminal and bridging hydride ligands interchange intramolecularly at a rate of 1 s(-1) at -40 °C. The compound reacts with D2 to afford [D1D](0), but not mixed isotopomers such as [H1D](0). The dihydride undergoes oxidation with Fc(+) under CO to give [1(CO)](+) and H2. Protonation in MeCN solution gives [H1(NCMe)](+) and H2. Carbonylation converts [H1H](0) into [1(CO)](0).

Synthesis and characterization of the platinum-substituted Keggin anion α-H2SiPtW11O40(4-).

Acidification of an aqueous solution of K8SiW11O39 and K2Pt(OH)6 to pH 4 followed by addition of excess tetramethylammonium (TMA) chloride yielded a solid mixture of TMA salts of H2SiPtW11O40(4-) (1) and SiW12O40(4-) (2). The former was separated from the latter by extraction into an aqueous solution and converted into tetra-n-butylammonium (TBA) and potassium salts TBA-1 and K-1. The α-H2SiPtW11O40(4-) was identified as a monosubstituted Keggin anion using elemental analysis, IR spectroscopy, X-ray crystallography, electrospray ionization mass spectrometry, (195)Pt NMR spectroscopy, (183)W NMR spectroscopy, and (183)W-(183)W 2D INADEQUATE NMR spectroscopy. Both TBA-1 and K-1 readily cocrystallized with their unsubstituted Keggin anion salts, TBA-2 and K-2, respectively, providing an explanation for the historical difficulty of isolating certain platinum-substituted heteropolyanions in pure form.

Unexpected transformations during pyrroloiminoquinone biosynthesis.

Pyrroloiminoquinone containing natural products have long been known for their biological activities. They are derived from tryptophan, but their biosynthetic pathways have remained elusive. Studies on the biosynthetic gene cluster (BGC) that produces the ammosamides revealed that the first step is attachment of Trp to the C-terminus of a scaffold peptide in an ATP and tRNA dependent manner catalyzed by a PEptide Amino-acyl tRNA ligase (PEARL). The indole of the Trp is then oxidized to a hydroxyquinone. We previously proposed a chemically plausible and streamlined pathway for converting this intermediate to the ammosamides using additional enzymes encoded in the BGC. In this study, we report the activity of four additional enzymes that show that the proposed pathway is incorrect and that Nature's route towards pyrroloiminoquinones is much more complicated. We demonstrate that, surprisingly, the amino groups in pyrroloiminoquinones are derived from three different sources, glycine, asparagine, and leucine, all introduced in a tRNA dependent manner. We also show that an FAD-dependent putative glycine oxidase is required for the process that incorporates the nitrogens from glycine and leucine, and that a quinone reductase is required for the incorporation of the asparagine. Additionally, we provide the first insights into the evolutionary origin of the PEARLs as well as related enzymes such as the glutamyl-tRNA dependent dehydratases involved in the biosynthesis of lanthipeptides and thiopeptides. These enzymes appear to all have descended from the ATP-GRASP protein family.

Biosynthesis of macrocyclic peptides with C-terminal ß-amino-α-keto acid groups by three different metalloenzymes.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new class involving modifications installed by a cytochrome P450, a multi-nuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes encoded by the BGC were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization with 2D-NMR and Marfey's method on the resulting RiPP demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C crosslink between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid while the methyltransferase acted on the ß-carbon of the α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configurations of the atropisomer that formed upon biaryl crosslinking. The conserved Cys residue in the precursor peptide was not modified as in all other characterized MNIO-containing BGCs; However, mutational analyses demonstrated that it was essential for the MNIO activity on the C-terminal Asp. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to discover new macrocyclic RiPPs and that RiPPs remain a significant source of previously undiscovered enzyme chemistry.

Biosynthesis of Macrocyclic Peptides with C-Terminal ß-Amino-α-keto Acid Groups by Three Different Metalloenzymes.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-l-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C cross-link between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the ß-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.