Fungal Indole Prenyltransferase Research Articles

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.

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.

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.

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.

Origin of product selectivity in a prenyl transfer reaction from the same intermediate: exploration of multiple FtmPT1-catalyzed prenyl transfer pathways.

FtmPT1 is a fungal indole prenyltransferase that catalyzes the reaction of tryptophan derivatives with dimethylallyl pyrophosphate to form various biologically active compounds. Herein, we describe detailed studies of FtmPT1 catalysis involving dimethylallyl pyrophosphate and Brevianamide F following the native pathway (yielding Tryprostatin B) and an alternate pathway observed in the Gly115Thr mutant of FtmPT1 yielding a novel cyclized product. Importantly, these two products arise from the same intermediate state, meaning that a step other than the cleavage of the dimethylallyl pyrophosphate (DMAPP; C–O) bond is differentiating between the two product reaction channels. From detailed potential of mean force (PMF) and two-dimensional PMF analyses, we conclude that the rate-limiting step is the cleavage of the C–O bond in DMAPP, while the deprotonation/cyclization step determines the final product distribution. Hence, in the case of FtmPT1, the optimization of the necessary catalytic machinery guides the generation of the final product after formation of the intermediate carbocation.

Structure and Catalytic Mechanism of a Cyclic Dipeptide Prenyltransferase with Broad Substrate Promiscuity

Fungal indole prenyltransferases (PTs) typically act on specific substrates, and they are able to prenylate their target compounds with remarkably high regio- and stereoselectivity. Similar to several indole PTs characterized to date, the cyclic dipeptide N-prenyltransferase (CdpNPT) is able to prenylate a range of diverse substrates, thus exhibiting an unusually broad substrate promiscuity. To define the structural basis for this promiscuity, we have determined crystal structures of unliganded CdpNPT and of a ternary complex of CdpNPT bound to (S)-benzodiazepinedione and thiolodiphosphate. Analysis of the structures reveals a limited number of specific interactions with (S)-benzodiazepinedione, which projects into a largely hydrophobic surface. This surface can also accommodate other substrates, explaining the ability of the enzyme to prenylate a range of compounds. The location of the bound substrates suggests a likely reaction mechanism for the conversion of (S)-benzodiazepinedione. Structure-guided mutagenesis experiments confirm that, in addition to (S)-benzodiazepinedione, CdpNPT can also act on (R)-benzodiazepinedione and several cyclic dipeptides, albeit with relaxed specificity. Finally, nuclear magnetic resonance spectroscopy demonstrates that CdpNPT is a C-3 reverse PT that catalyzes the formation of C-3β prenylated indolines from diketopiperazines of tryptophan-containing cyclic dipeptides.

Identification of a brevianamide F reverse prenyltransferase BrePT from Aspergillus versicolor with a broad substrate specificity towards tryptophan-containing cyclic dipeptides

A putative brevianamide F reverse prenyltransferase gene brePT was amplified from Aspergillus versicolor NRRL573 by using primers deduced from its orthologue notF in Aspergillus sp. MF297-2 and overexpressed in Escherichia coli. The soluble His-tagged protein BrePT was purified to near homogeneity and assayed with tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. BrePT showed much higher flexibility towards its aromatic substrates than NotF and accepted all of the 14 tested tryptophan-containing cyclic dipeptides. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific reverse prenylation at C2 of the indole nucleus. K(M) values of BrePT were determined for its putative substrates brevianamide F and DMAPP at 32 and 98 μM, respectively. Average turnover number (k (cat)) at 0.4 s⁻¹ was calculated from kinetic data of brevianamide F and DMAPP. K(M) values in the range of 0.082-2.9 mM and k (cat) values from 0.003 to 0.15 s⁻¹ were determined for other 11 cyclic dipeptides. Similar to known fungal indole prenyltransferases, BrePT did not accept geranyl or farnesyl diphosphate as prenyl donor for its prenylation.

Prenylation of a Nonaromatic Carbon of Indolylbutenone by a Fungal Indole Prenyltransferase

FtmPT1 from Aspergillus fumigatus is a fungal indole prenyltransferase (PT) that normally catalyzes the regiospecific prenylation of brevianamide F (cyclo-L-Trp-L-Pro) at the C-2 position of the indole ring with dimethylallyl diphosphate (DMAPP). Interestingly, FtmPT1 exhibited remarkable substrate tolerance and accepted (E)-4-(1H-indol-3-yl)but-3-en-2-one (1) as a substrate to produce an unnatural novel α-prenylindolylbutenone (1a). This is the first demonstration of the prenylation of a nonaromatic carbon of the acceptor substrate by a fungal indole PT.

Substrate promiscuity of secondary metabolite enzymes: prenylation of hydroxynaphthalenes by fungal indole prenyltransferases

Fungal prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily share no sequence, but structure similarity with the prenyltransferases of the CloQ/NphB group. The members of the DMATS superfamily have been reported to catalyze different prenylations of diverse indole or tyrosine derivatives, while some members of the CloQ/NphB group used hydroxynaphthalenes as prenylation substrates. In this study, we report for the first time the prenylation of hydroxynaphthalenes by the members of the DMATS superfamily. Three tryptophan-containing cyclic dipeptide prenyltransferases (AnaPT, CdpNPT and CdpC3PT), one tryptophan C7-prenyltransferase and one tyrosine O-prenyltransferase (SirD) were incubated with naphthalene and 11 derivatives. The enzyme activity and preference of the tested prenyltransferases towards hydroxynaphthalenes differed clearly from each other. For an accepted substrate, however, different enzymes produced usually the same major prenylation product, i.e. with a regular C-prenyl moiety at para- or ortho-position to a hydroxyl group. Regularly, O-prenylated and diprenylated derivatives were also identified as enzyme products of substrates with low conversion rates and regioselectivity. This was unequivocally proven by mass spectrometry and nuclear magnetic resonance analyses. The K (M) values and turnover numbers (k (cat)) of the enzymes towards selected hydroxynaphthalenes were determined to be in the range of 0.064-2.8 mM and 0.038-1.30 s(-1), respectively. These data are comparable to those obtained using indole derivatives. The results presented in this study expanded the potential usage of the members of the DMATS superfamily for production of prenylated derivatives including hydroxynaphthalenes.

Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans.

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.

Structure−Function Analysis of an Enzymatic Prenyl Transfer Reaction Identifies a Reaction Chamber with Modifiable Specificity

Fungal indole prenyltransferases participate in a multitude of biosynthetic pathways. Their ability to prenylate diverse substrates has attracted interest for potential use in chemoenzymatic synthesis. The fungal indole prenyltransferase FtmPT1 catalyzes the prenylation of brevianamide F in the biosynthesis of fumitremorgin-type alkaloids, which show diverse pharmacological activities and are promising candidates for the development of antitumor agents. Here, we report crystal structures of unliganded Aspergillus fumigatus FtmPT1 as well as of a ternary complex of FtmPT1 bound to brevianamide F and an analogue of its isoprenoid substrate dimethylallyl diphosphate. FtmPT1 assumes a rare α/β-barrel fold, consisting of 10 circularly arranged β-strands surrounded by α-helices. Catalysis is performed in a hydrophobic reaction chamber at the center of the barrel. In combination with mutagenesis experiments, our analysis of the liganded and unliganded structures provides insight into the mechanism of catalysis and the determinants of regiospecificity. Sequence conservation of key features indicates that all fungal indole prenyltransferases possess similar active site architectures. However, while the dimethylallyl diphosphate binding site is strictly conserved in these enzymes, subtle changes in the reaction chamber likely allow for the accommodation of diverse aromatic substrates for prenylation. In support of this concept, we were able to redirect the regioselectivity of FtmPT1 by a single mutation of glycine 115 to threonine. This finding provides support for a potential use of fungal indole prenyltransferases as modifiable bioreactors that can be engineered to catalyze highly specific prenyl transfer reactions.

Biochemical Characterization of a Novel Indole Prenyltransferase from Streptomyces sp. SN-593

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (-)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min(-1) microM(-1)) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, DeltaiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.

Increasing structure diversity of prenylated diketopiperazine derivatives by using a 4-dimethylallyltryptophan synthase

To create structural diversity of prenylated diketopiperazine derivatives, acceptance of cyclic dipeptides was tested using FgaPT2, a prenyltransferase from Aspergillus fumigatus, which catalyses the conversion of L: -tryptophan to 4-dimethylallyl-L: -tryptophan. It could be shown that seven tryptophan-containing cyclic dipeptides were accepted by FgaPT2 at high protein concentrations and regiospecifically converted to their C4 prenylated derivatives. The structures of the enzymatic products were elucidated by NMR and LC-MS analyses. This substrate promiscuity of a dimethylallyltryptophan synthase towards cyclic dipeptides increases the potential of the fungal indole prenyltransferases as tools for the production of biologically active compounds.