Indole Prenyltransferases Research Articles

Selective Late-Stage Functionalization of Tryptophan-Containing Peptides To Facilitate Bioorthogonal Tetrazine Ligation.

Site-selective modification of complex peptides and the functionalization of their C-H bonds hold great promise for expanding their use in therapeutics and biomedical research. Herein, we leverage the power of late-stage chemoenzymatic catalysis using an indole prenyltransferase (IPT) enzyme and alkyl diphosphates to specifically modify the indole ring of tryptophan in clinically relevant peptides. Furthermore, the installed handle enables bioorthogonal click chemistry through an inverse electron-demand Diels-Alder (IEDDA) reaction with a biotin-conjugated tetrazine probe.

Prenylation of dimeric cyclo-l-Trp-l-Trp by the promiscuous cyclo-l-Trp-l-Ala prenyltransferase EchPT1

Prenyltransferases (PTs) from the dimethylallyl tryptophan synthase (DMATS) superfamily are known as efficient biocatalysts and mainly catalyze regioselective Friedel-Crafts alkylation of tryptophan and tryptophan-containing cyclodipeptides (CDPs). They can also use other unnatural aromatic compounds as substrates and play therefore a pivotal role in increasing structural diversity and biological activities of a broad range of natural and unnatural products. In recent years, several prenylated dimeric CDPs have been identified with wide range of bioactivities. In this study, we demonstrate the production of prenylated dimeric CDPs by chemoenzymatic synthesis with a known promiscuous enzyme EchPT1, which uses cyclo-l-Trp-l-Ala as natural substrate for reverse C2-prenylation. High product yields were achieved with EchPT1 for C3-N1′ and C3-C3′ linked dimers of cyclo-l-Trp-l-Trp. Isolation and structural elucidation confirmed the product structures to be reversely C19/C19′-mono- and diprenylated cyclo-l-Trp-l-Trp dimers. Our study provides an additional example for increasing structural diversity by prenylation of complex substrates with known biosynthetic enzymes.Key points• Chemoenzymatic synthesis of prenylated cyclo-l-Trp-l-Trp dimers• Same prenylation pattern and position for cyclodipeptides and their dimers.• Indole prenyltransferases such as EchPT1 can be widely used as biocatalysts.Graphical

Unlocking New Prenylation Modes: Azaindoles as a New Substrate Class for Indole Prenyltransferases.

Aza-substitution, the replacement of aromatic CH groups with nitrogen atoms, is an established medicinal chemistry strategy for increasing solubility, but current methods of accessing functionalized azaindoles are limited. In this work, indole-alkylating aromatic prenyltransferases (PTs) were explored as a strategy to directly functionalize azaindole-substituted analogs of natural products. For this, a series of aza-l-tryptophans (Aza-Trp) featuring N-substitution of every aromatic CH position of the indole ring and their corresponding cyclic Aza-l-Trp-l-proline dipeptides (Aza-CyWP), were synthesized as substrate mimetics for the indole-alkylating PTs FgaPT2, CdpNPT, and FtmPT1. We then demonstrated most of these substrate analogs were accepted by a PT, and the regioselectivity of each prenylation was heavily influenced by the position of the N-substitution. Remarkably, FgaPT2 was found to produce cationic N-prenylpyridinium products, representing not only a new substrate class for indole PTs but also a previously unobserved prenylation mode. The discovery that nitrogenous indole bioisosteres can be accepted by PTs thus provides access to previously unavailable chemical space in the search for bioactive indolediketopiperazine analogs.

Structure-guided Mutagenesis Reveals the Catalytic Residue that Controls the Regiospecificity of C6-Indole Prenyltransferases.

Indole is a significant structural moiety and functionalization of the C-H bond in indole-containing molecules expands their chemical space, and modifies their properties and/or activities. Indole prenyltransferases (IPTs) catalyze the direct regiospecific installation of prenyl, C5 carbon units, on indole-derived compounds. IPTs have shown relaxed substrate flexibility enabling them to be used as tools for indole functionalization. However, the mechanism by which certain IPTs target a specific carbon position is not fully understood. Herein, we use structure-guided site-directed mutagenesis, in vitro enzymatic reactions, kinetics and structural-elucidation of analogs to verify the key catalytic residues that control the regiospecificity of all characterized regiospecific C6 IPTs. Our results also demonstrate that substitution of PriB_His312 to Tyr leads to the synthesis of analogs prenylated at different positions than C6. This work contributes to understanding of how certain IPTs can access a challenging position in indole-derived compounds.

Catalytic potential of a fungal indole prenyltransferase toward β-carbolines, harmine and harman, and their prenylation effects on antibacterial activity

The prenylation of compounds has attracted much attention, since it often adds bioactivity to non-prenylated compounds. We employed an enzyme assay with CdpNPT, an indole prenyltransferase from Aspergillus fumigatus with two naturally occurring β-carbolines, harmine (3) and harman (4) as prenyl acceptors, in the presence of dimethylallyl diphosphate (DMAPP) as the prenyl donor. The enzyme accepted these two prenyl acceptor substrates to produce 6-(3',3'-dimethylallyl)harmine (5) from 3 and 9-(3',3'-dimethylallyl)harman (6) and 6-(3',3'-dimethylallyl)harman (7) from 4. The X-ray crystal structure analysis of the CdpNPT (38-440) truncated mutant complexed with 4, and docking simulation studies of DMAPP to the crystal structure of the CdpNPT (38-440) mutant, suggested that CdpNPT could employ the two-step prenylation mechanism to produce 7, while the enzyme produced 6 with either one- or two-step prenylation mechanisms. Furthermore, the antibacterial assays revealed that the 3',3'-dimethylallylation of 3 and 4, as well as harmol (1), at C-6 enhanced the activities against Staphylococcus aureus and Bacillus subtilis.

Enzymatic formation of a prenyl β-carboline by a fungal indole prenyltransferase.

CdpNPT from Aspergillus fumigatus is a fungal indole prenyltransferase (IPT) with remarkable substrate promiscuity to generate prenylated compounds. Our first investigation of the catalytic potential of CdpNPT against a β-carboline, harmol (1), revealed that the enzyme also accepts 1 as the prenyl acceptor with dimethylallyl diphosphate (DMAPP) as the prenyl donor and selectively prenylates the C-6 position of 1 by the "regular-type" dimethylallylation to produce 6-(3-dimethylallyl)harmol (2). Furthermore, our X-ray crystal structure analysis of the C-His6-tagged CdpNPT (38-440) truncated mutant complexed with 1 and docking studies of DMAPP to the crystal structure of the CdpNPT (38-440) mutant suggested that CdpNPT could employ the two-step prenylation system to produce 2.

Late-Stage Chemoenzymatic Installation of Hydroxy-Bearing Allyl Moiety on the Indole Ring of Tryptophan-Containing Peptides.

The late‐stage functionalization of indole‐ and tryptophan‐containing compounds with reactive moieties facilitates downstream diversification and leads to changes in their biological properties. Here, the synthesis of two hydroxy‐bearing allyl pyrophosphates is described. A chemoenzymatic method is demonstrated which uses a promiscuous indole prenyltransferase enzyme to install a dual reactive hydroxy‐bearing allyl moiety directly on the indole ring of tryptophan‐containing peptides. This is the first report of late‐stage indole modifications with this reactive group.

Bioinspired Brønsted Acid-Promoted Regioselective Tryptophan Isoprenylations.

Tryptophan-containing isoprenoid indole alkaloid natural products are well known for their intricate structural architectures and significant biological activities. Nature employs dimethylallyl tryptophan synthases (DMATSs) or aromatic indole prenyltransferases (iPTs) to catalyze regio- and stereoselective prenylation of l-Trp. Regioselective synthetic routes that isoprenylate cyclo-Trp-Trp in a 2,5-diketopiperazine (DKP) core, in a desymmetrizing manner, are nonexistent and are highly desirable. Herein, we present an elaborate report on Brønsted acid-promoted regioselective tryptophan isoprenylation strategy, applicable to both the monomeric amino acid and its dimeric l-Trp DKP. This report outlines a method that regio- and stereoselectively increases sp3 centers of a privileged bioactive core. We report on conditions involving screening of Brønsted acids, their conjugate base as salt, solvent, temperature, and various substrates with diverse side chains. Furthermore, we extensively delineate effects on regio- and stereoselection of isoprenylation and their stereochemical confirmation via NMR experiments. Regioselectively, the C3-position undergoes normal-isoprenylation or benzylation and forms exo-ring-fused pyrroloindolines selectively. Through appropriate prenyl group migrations, we report access to the bioactive tryprostatin alkaloids, and by C3-normal-farnesylation, we access anticancer drimentines as direct targets of this method. The optimized strategy affords iso-tryprostatin B-type products and predrimentine C with 58 and 55% yields, respectively. The current work has several similarities to biosynthesis, such as—reactions can be performed on unprotected substrates, conditions that enable Brønsted acid promotion, and they are easy to perform under ambient conditions, without the need for stoichiometric levels of any transition metal or expensive ligands.

Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by CymD.

Indole prenyltransferases catalyze the prenylation of l-tryptophan (l-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from Salinispora arenicola catalyzes the reverse N1 prenylation of l-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation. To better understand the structural basis of selective indole N-alkylation reactions in biology, we have determined the X-ray crystal structures of CymD, the CymD-l-Trp complex, and the CymD-l-Trp-DMSPP complex (DMSPP is dimethylallyl S-thiolodiphosphate, an unreactive analogue of DMAPP). The orientation of l-Trp with respect to DMSPP reveals how the active site contour of CymD serves as a template to direct the reverse prenylation of the indole nitrogen. Comparison to PriB, a C6 bacterial indole prenyltransferase, offers further insight regarding the structural basis of regioselective indole prenylation. Isothermal titration calorimetry measurements indicate a synergistic relationship between l-Trp and DMSPP binding. Finally, activity assays demonstrate the selectivity of CymD for l-Trp and indole as prenyl acceptors. Collectively, these data establish a foundation for understanding and engineering the regioselectivity of indole prenylation by members of the prenyltransferase protein family.

Structural insight into a novel indole prenyltransferase in hapalindole-type alkaloid biosynthesis

FamD1 is a novel CloQ/NphB-family indole prenyltransferase which involves in hapalindole-type alkaloid biosynthesis. Here the native FamD1 structure and three protein-ligand complexes are analyzed to investigate the molecular basis of substrate binding and catalysis. FamD1 adopts a typical ABBA architecture of aromatic prenyltransferase, in which the substrate-binding chamber is found in the central β-barrel. The indole-containing acceptor substrate is bound adjacent to the prenyl donor. Based on the complex structures, a catalytic mechanism of FamD1 is proposed. Functional implications on the sister enzyme FamD2 are also discussed.

Mechanism of Formation of the Nonstandard Product in the Prenyltransferase Reaction of the G115T Mutant of FtmPT1: A Case of Reaction Dynamics Calling the Shots?

FtmPT1 is a fungal indole prenyltransferase that affords Tryprostatin B from Brevianamide F and dimethylallyl pyrophosphate; however, when a single residue in the active site is mutated (Gly115Thr), a novel five-membered ring compound is obtained as the major product with Tryprostatin B as the minor product. Herein, we describe detailed studies of the catalysis of the Gly115Thr mutant of FtmPT1 with a focus on the observed regioselectivity of the reaction. We employ one- and two-dimensional potential of mean force simulations to explore the catalytic mechanism, along with molecular dynamics simulations exploring the reaction dynamics of the prenyl transfer reaction. Single-point electronic structure calculations were also used to explore the performance of the self-consistent charge density functional tight-binding method to model specific reaction steps. Importantly, we observe that the two reaction pathways have comparable activation parameters and propose that the origin of the novel product is predicated, at least in part, on the topology of the potential energy surface as revealed by reaction dynamics studies.

Saturation mutagenesis on Tyr205 of the cyclic dipeptide C2-prenyltransferase FtmPT1 results in mutants with strongly increased C3-prenylating activity.

The fungal indole prenyltransferase FtmPT1 is involved in the biosynthesis of fumitremorgins and catalyzes, in the presence of dimethylallyl diphosphate, a predominant regular prenylation of cyclo-L-Trp-L-Pro (brevianamide F) at position C-2 of the indole nucleus. Analysis of the substrate-bound structure of FtmPT1 revealed that brevianamide F forms a hydrogen bond via its carbonyl oxygen in the diketopiperazine moiety with the hydroxyl group of Tyr205 near the center of the prenyltransferase (PT) barrel. In this study, Tyr205 was mutated to 19 other proteinogenic amino acids by one-step site-directed mutagenesis. The obtained mutants were assayed in the presence of dimethylallyl diphosphate with brevianamide F. The enzyme products were isolated on HPLC and their structures were elucidated by NMR and MS analyses. Mutation of Tyr205 to Phe or Met did not change the behavior of FtmPT1 significantly, with regularly C2-prenylated brevianamide F as the predominant product. Interestingly, 15 of the obtained mutants also produced regularly C3-prenylated brevianamide F, with relative yields between 33 and 110% of those of the regularly C2-prenylated derivatives. Among them, Y205C, Y205L, Y205N, Y205I, and Y205S showed similar brevianamide F consumption. Y205H, Y205Q, Y205V, Y205G, and Y205E showed activities between 47 and 77% of that of the wild type. These results provide a solid basis for the construction of a brevianamide F regular C3-prenyltransferase by site-directed mutagenesis. Assaying stereoisomers of brevianamide F, cyclo-D-Trp-D-Pro, cyclo-L-Trp-D-Pro, and cyclo-D-Trp-L-Pro, with two selected mutants Y205N and Y205L resulted in the formation of reversely C3-prenylated derivatives as predominant products, being in sharp contrast to their regularly C2- and C3-prenylated derivatives with cyclo-L-Trp-L-Pro.

Metabolomics-Driven Discovery of a Prenylated Isatin Antibiotic Produced by Streptomyces Species MBT28.

Actinomycetes are a major source of antimicrobials, anticancer compounds, and other medically important products, and their genomes harbor extensive biosynthetic potential. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known molecules. Here we report the identification of a previously unidentified isatin-type antibiotic produced by Streptomyces sp. MBT28, following a strategy based on NMR-based metabolomics combined with the introduction of streptomycin resistance in the producer strain. NMR-guided isolation by tracking the target proton signal resulted in the characterization of 7-prenylisatin (1) with antimicrobial activity against Bacillus subtilis. The metabolite-guided genome mining of Streptomyces sp. MBT28 combined with proteomics identified a gene cluster with an indole prenyltransferase that catalyzes the conversion of tryptophan into 7-prenylisatin. This study underlines the applicability of NMR-based metabolomics in facilitating the discovery of novel antibiotics.

Complementary Flavonoid Prenylations by Fungal Indole Prenyltransferases.

Flavonoids are found mainly in plants and exhibit diverse biological and pharmacological activities, which can often be enhanced by prenylations. In plants, such reactions are catalyzed by membrane-bound prenyltransferases. In this study, the prenylation of nine flavonoids from different classes by a soluble fungal prenyltransferase (AnaPT) involved in the biosynthesis of the prenylated indole alkaloid acetylaszonalenin is demonstrated. The behavior of AnaPT toward flavonoids regarding substrate acceptance and prenylation positions clearly differs from that of the indole prenyltransferase 7-DMATS. The two enzymes are therefore complementary in flavonoid prenylations.

ChemInform Abstract: Regioselective Cope Rearrangement and Prenyl Transfers on Indole Scaffold Mimicking Fungal and Bacterial Dimethylallyltryptophan Synthases.

Aromatic prenyltransferases are an actively mined enzymatic class whose biosynthetic repertoire is growing. Indole prenyltransferases catalyze the formation of a diverse set of prenylated tryptophan and diketopiperazines, leading to the formation of fungal toxins with prolific biological activities. At a fundamental level, the mechanism of C4-prenylation of l-tryptophan recently has surfaced to engage a debate between a “direct” electrophilic alkylation mechanism (for wt DMATS and FgaPT2) versus an indole C3–C4 “Cope” rearrangement followed by rearomatization (for mutant FgaPT2). Herein we provide the first series of regioselectively tunable conditions for a Cope rearrangement between C3 and C4 positions. Biomimetic conditions are reported that effect a [3,3]-sigmatropic shift whose two-step process is interrogated for intramolecularity and rate-limiting general base-promoted mechanism. Solvent polarity serves a crucial role in changing the regioselectivity, resulting in sole [1,3]-shifts under decalin. A...

Friedel-crafts alkylation of acylphloroglucinols catalyzed by a fungal indole prenyltransferase.

Naturally occurring prenylated acylphloroglucinol derivatives are plant metabolites with diverse biological and pharmacological activities. Prenylation of acylphloroglucinols plays an important role in the formation of these intriguing natural products and is catalyzed in plants by membrane-bound enzymes. In this study, we demonstrate the prenylation of such compounds by a soluble fungal prenyltransferase AnaPT involved in the biosynthesis of prenylated indole alkaloids. The observed activities of AnaPT toward these substrates are much higher than that of a microsomal fraction containing an overproduced prenyltransferase from the plant hop.

Tyrosine O-prenyltransferases TyrPT and SirD displaying similar behavior toward unnatural alkyl or benzyl diphosphate as their natural prenyl donor dimethylallyl diphosphate.

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily are involved in the biosynthesis of secondary metabolites and contribute as modification enzymes significantly to structural diversity of natural products. They show usually broad specificity toward their aromatic substrates with regiospecific prenylations on aromatic rings. However, most members of this superfamily exhibit a high specificity toward their prenyl donors and usually accept exclusively dimethylallyl diphosphate (DMAPP). Recently, several indole prenyltransferases from this family were also demonstrated to accept unnatural DMAPP analogs such as methylallyl, 2-pentenyl and benzyl diphosphate for alkylation, or benzylation of the indole ring. Partial or complete shift of the substitution position was observed for these enzymes. In this study, we report the acceptance of these DMAPP analogs by two tyrosine O-prenyltransferases TyrPT from Aspergillus niger and SirD from Leptosphaeria maculans for alkylation or benzylation of tyrosine and derivatives. NMR and mass spectrometry (MS) analyses of nine isolated enzyme products confirmed the regiospecific O- or N-alkylation or benzylation at position C-4 of the aromatic ring, which is the same prenylation position of these enzymes in the presence of DMAPP.

Mechanistic studies on the indole prenyltransferases.

Covering: up to 2014. Prenylated indole alkaloids comprise a large and structurally diverse family of natural products that often display potent biological activities. In recent years a large family of prenyltransferases that install prenyl groups onto the indole core have been discovered. While the vast majority of these enzymes are evolutionarily related and share a common protein fold, they are remarkably versatile in their ability to catalyze reverse and normal prenylations at all positions on the indole ring. This highlight article will focus on recent studies of the mechanisms utilized by indole prenyltransferases. While all of the prenylation reactions may follow a direct electrophilic aromatic substitution mechanism, studies of structure and reactivity suggest that in some cases prenylation may first occur at the nucleophilic C-3 position, and subsequent rearrangements then generate the final product.

Biological matching of chemical reactivity: pairing indole nucleophilicity with electrophilic isoprenoids.

The indole side chain of tryptophan has latent nucleophilic reactivity at both N1 and all six (nonbridgehead) carbons, which is not generally manifested in post-translational reactions of proteins. On the other hand, all seven positions can be prenylated by the primary metabolite Δ(2)-isopentenyl diphosphate by dimethyallyl transferase (DMATs) family members as initial steps in biosynthetic pathways to bioactive fungal alkaloids including ergots and tremorgens. These are formulated as regioselective capture of isopentenyl allylic cationic transition states by the indole side chain as a nucleophile. The balance of regiospecificity and promiscuity among these indole prenyltransferases continues to raise questions about possible Cope and azaCope rearrangements of nascent products. In addition to these two electron reaction manifolds, there is evidence for one electron reaction manifolds in indole ring biosynthetic functionalization.

Regioselective cope rearrangement and prenyl transfers on indole scaffold mimicking fungal and bacterial dimethylallyltryptophan synthases.

Aromatic prenyltransferases are an actively mined enzymatic class whose biosynthetic repertoire is growing. Indole prenyltransferases catalyze the formation of a diverse set of prenylated tryptophan and diketopiperazines, leading to the formation of fungal toxins with prolific biological activities. At a fundamental level, the mechanism of C4-prenylation of l-tryptophan recently has surfaced to engage a debate between a "direct" electrophilic alkylation mechanism (for wt DMATS and FgaPT2) versus an indole C3-C4 "Cope" rearrangement followed by rearomatization (for mutant FgaPT2). Herein we provide the first series of regioselectively tunable conditions for a Cope rearrangement between C3 and C4 positions. Biomimetic conditions are reported that effect a [3,3]-sigmatropic shift whose two-step process is interrogated for intramolecularity and rate-limiting general base-promoted mechanism. Solvent polarity serves a crucial role in changing the regioselectivity, resulting in sole [1,3]-shifts under decalin. An intermolecular variant is also reported that effectively prenylates the C3 position of l-tryptophan, resulting in products that mimic the structures accessed by bacterial indole prenyltransferases. We report an elaborate investigation that includes screening various substituents and measuring steric and electronic effects and stereoselectivity with synthetically useful transformations.