RESUMEN
A conserved two-gene cassette in fungi was discovered by genome mining, which encodes a UbiA family intramembrane prenyltansferase (VviA) and a haloacid dehalogenase-like hydrolase family terpene cyclase (VviB), respectively. A series of in vivo and in vitro investigations revealed that VviA exclusively uses VviB-synthesized drim-8-ene diphosphate (cyclo-farnesyl diphosphate) as the native prenyl donor to catalyze prenylation on d-mannitol, showcasing a previously unidentified function of UbiA-type prenyltransferases and a new prenylation manner in fungi.
Asunto(s)
Dimetilaliltranstransferasa , Prenilación , Estructura Molecular , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Hongos/química , Hongos/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismoRESUMEN
Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by 1H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor's biological activity.
Asunto(s)
Dimetilaliltranstransferasa , Mononucleótido de Flavina , Prenilación , Mononucleótido de Flavina/metabolismo , Mononucleótido de Flavina/química , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Riboflavina/biosíntesis , Riboflavina/análogos & derivados , Riboflavina/metabolismo , Riboflavina/química , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/química , Catálisis , Compuestos Alílicos/metabolismo , Compuestos Alílicos/química , Escherichia coli/metabolismo , Escherichia coli/genética , Carboxiliasas , HemiterpenosRESUMEN
Prenylation of amino acids is a critical step for synthesizing building blocks of prenylated alkaloid family natural products, where the corresponding prenyltransferase that catalyzes prenylation on free l-histidine (l-His) has not yet been identified. Here, we first discovered and characterized a prenyltransferase FunA from the antifungal agent fungerin pathway that efficiently performs C4-dimethylallylation on l-His. Crystal structure-guided engineering of the prenyl-binding pocket of FunA, a single M181A mutation, successfully converted it into a C4-geranyltransferase. Furthermore, FunA and its variant FunA-M181A show broad substrate promiscuity toward substrates that vary in substituents of the imidazole ring. Our work furthers our knowledge of free amino acid prenyltransferase and expands the arsenal of alkylation biocatalysts for imidazole-containing small molecules.
Asunto(s)
Dimetilaliltranstransferasa , Histidina , Histidina/química , Histidina/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Ingeniería de Proteínas , Modelos Moleculares , Especificidad por Sustrato , Imidazoles/química , Imidazoles/metabolismoRESUMEN
New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.
Asunto(s)
Transferasas Alquil y Aril , Dimetilaliltranstransferasa , Diterpenos , Streptomyces , Diterpenos/química , Diterpenos/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Streptomyces/enzimología , Streptomyces/química , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Estructura Molecular , Ciclización , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , BiotransformaciónRESUMEN
Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.
Asunto(s)
Transferasas Alquil y Aril , Microscopía por Crioelectrón , Penicillium , Penicillium/enzimología , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/aislamiento & purificación , Microscopía por Crioelectrón/métodos , Diterpenos/metabolismo , Diterpenos/química , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Dimetilaliltranstransferasa/aislamiento & purificaciónRESUMEN
Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.
Asunto(s)
Transferasas Alquil y Aril , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Diterpenos/metabolismo , Diterpenos/química , Pruebas de Enzimas/métodos , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Poliisoprenilo/química , CiclizaciónRESUMEN
Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30â nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.
Asunto(s)
Prenilación , Ligandos , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Compuestos Macrocíclicos/metabolismo , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/antagonistas & inhibidores , Prenilación de ProteínaRESUMEN
Farnesylated chalcones were favored by researchers due to their different biological activities. However, only five naturally occurring farnesylated chalcones were described in the literature until now. Here, the farnesylation of six chalcones by the Aspergillus terreus aromatic prenyltransferase AtaPT was reported. Fourteen monofarnesylated chalcones (1F1-1F5, 2F1-2F3, 3F1, 3F2, 4F1, 4F2, 5F1, 6F1, and 6F2) and a difarnesylated product (2F3) were obtained, enriching the diversity of natural farnesylated chalcones significantly. Ten of them are C-farnesylated products, which complement O-farnesylated chalcones by chemical synthesis. Fourteen products have not been reported prior to this study. Nine of the produced compounds (1F2-1F5, 2F1-2F3, 5F1, and 6F1) exhibited inhibitory effect on α-glucosidase with IC50 values ranging from 24.08 ± 1.44 to 190.0 ± 0.28 µM. Among them, compounds 2F3 with IC50 value at 24.08 ± 1.44 µM and 1F4 with IC50 value at 30.09 ± 0.59 µM showed about 20 times stronger than the positive control acarbose with an IC50 at 536.87 ± 24.25 µM in α-glucosidase inhibitory assays.
Asunto(s)
Aspergillus , Chalconas , Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/antagonistas & inhibidores , Chalconas/química , Chalconas/farmacología , Chalconas/metabolismo , Aspergillus/enzimología , Aspergillus/química , Estructura Molecular , Prenilación , Relación Estructura-Actividad , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , alfa-Glucosidasas/metabolismo , alfa-Glucosidasas/química , Relación Dosis-Respuesta a DrogaRESUMEN
UbiA-type prenyltransferases (PTases) are significant enzymes that lead to structurally diverse meroterpenoids. Herein, we report the identification and characterization of an undescribed UbiA-type PTase, FtaB, that is responsible for the farnesylation of indole-containing diketopiperazines (DKPs) through genome mining. Heterologous expression of the fta gene cluster and non-native pathways result in the production of a series of new C2-farnesylated DKPs. This study broadens the reaction scope of UbiA-type PTases and expands the chemical diversity of meroterpenoids.
Asunto(s)
Dicetopiperazinas , Dimetilaliltranstransferasa , Prenilación , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Dicetopiperazinas/química , Dicetopiperazinas/metabolismo , Estructura Molecular , Familia de MultigenesRESUMEN
CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic acid (OA) and geranyl diphosphate (GPP). CsPT4 has a catalytic potential to produce a variety of CBGA analogs via regioselective C-prenylation of aromatic substrates having resorcylic acid skeletons including bibenzyl 2,4-dihydroxy-6-phenylethylbenzoic acid (DPA). In this study, we further investigated the substrate specificity of CsPT4 using phlorocaprophenone (PCP) and 2',4',6'-trihydroxydihydrochalcone (THDC), the isomers of OA and DPA, respectively, and demonstrated that CsPT4 catalyzed both C-prenylation and O-prenylation reactions on PCP and THDC that share acylphloroglucinol substructures. Interestingly, the kinetic parameters of CsPT4 for these substrates differed depending on whether they underwent C-prenylation or O-prenylation, suggesting that this enzyme utilized different substrate-binding modes suitable for the respective reactions. Aromatic prenyltransferases that catalyze O-prenylation are rare in the plant kingdom, and CsPT4 was notable for altering the reaction specificity between C- and O-prenylations depending on the skeletons of aromatic substrates. We also demonstrated that enzymatically synthesized geranylated acylphloroglucinols had potent antiausterity activity against PANC-1 human pancreatic cancer cells, with 4'-O-geranyl THDC being the most effective. We suggest that CsPT4 is a valuable catalyst to generate biologically active C- and O-prenylated molecules that could be anticancer lead compounds.
Asunto(s)
Cannabis , Dimetilaliltranstransferasa , Humanos , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismo , Prenilación , Catálisis , Especificidad por SustratoRESUMEN
Covering: 2009 up to August 2023Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.
Asunto(s)
Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/química , Prenilación , Hongos/metabolismoRESUMEN
Post-translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing genome mining methods combined with AlphaFold2-based predictions of protein interactions, PalQ , a prenyltransferase responsible for the tryptophan prenylation of RiPPs produced by Paenibacillus alvei, is identified. PalQ differs from cyanobactin prenyltransferases because of its evolutionary relationship to isoprene synthases, which enables PalQ to transfer extended prenyl chains to the indole C3 position. This prenylation introduces structural diversity to the tryptophan side chain and also leads to conformational dynamics in the peptide backbone, attributed to the cis/trans isomerization that arises from the formation of a pyrrolidine ring. Additionally, PalQ exhibited pronounced positional selectivity for the C-terminal tryptophan. Such enzymatic characteristics offer a toolkit for peptide therapeutic lipidation.
Asunto(s)
Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/genética , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismo , Triptófano/química , Triptófano/genética , Triptófano/metabolismo , Prenilación , Procesamiento Proteico-Postraduccional , Péptidos/metabolismoRESUMEN
Prenyltransferases in cyanobactin biosynthesis are of growing interest as peptide alkylation biocatalysts, but their prenylation modes characterized so far have been limited to dimethylallylation (C5) or geranylation (C10). Here we engaged in structure-guided engineering of the prenyl-binding pocket of a His-C2-geranyltransferase LimF to modulate its prenylation mode. Contraction of the pocket by a single mutation led to a His-C2-dimethylallyltransferase. More importantly, pocket expansion by a double mutation successfully repurposed LimF for farnesylation (C15), which is an unprecedented mode in this family. Furthermore, the obtained knowledge of the essential residues to construct the farnesyl-binding pocket has allowed for rational design of a Tyr-O-farnesyltransferase by a triple mutation of a Tyr-O-dimethylallyltransferase PagF. These results provide an approach to manipulate the prenyl specificity of cyanobactin prenyltransferases, broadening the chemical space covered by this class of enzymes and expanding the toolbox of peptide alkylation biocatalysts.
Asunto(s)
Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/química , Péptidos Cíclicos , Prenilación , Péptidos/química , Especificidad por SustratoRESUMEN
NphB is an aromatic prenyltransferase with high promiscuity for phenolics including flavonoids, isoflavonoids, and plant polyketides. It has been demonstrated that cannabigerolic acid is successfully formed by the reaction catalysed by NphB using geranyl diphosphate and olivetolic acid as substrates. In this study, the substrate specificity of NphB was further determined by using olivetolic acid derivatives as potential substrates for the formation of new synthetic cannabinoids. The derivatives differ in the hydrocarbon chain attached to C6 of the core structure. We performed in silico experiments, including docking of olivetolic acid derivatives, to identify differences in their binding modes. Substrate acceptance was predicted. Based on these results, a library of olivetolic acid derivatives was constructed and synthesized by using different organic synthetic routes. Conversion was monitored in inâ vitro assays with purified NphB versions. For the substrates leading to a high conversion olivetolic acid-C8, olivetolic acid-C2 and 2-benzyl-4,6-dihydroxybenzoic acid, the products were further elucidated and identified as cannbigerolic acid derivatives. Therefore, these substrates show potential to be adapted in cannabinoid biosynthesis.
Asunto(s)
Cannabinoides , Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/química , Cannabinoides/metabolismo , Salicilatos/metabolismo , Especificidad por SustratoRESUMEN
The UbiX/UbiD system is widespread in microbes and responsible for the reversible decarboxylation of unsaturated carboxylic acids. The UbiD enzyme catalyzes this unusual reaction using a prenylated flavin (prFMN) as cofactor, the latter formed by the flavin prenyltransferase UbiX. A detailed picture of the biochemistry of flavin prenylation, oxidative maturation, and covalent catalysis underpinning reversible decarboxylation is emerging. This reveals the prFMN cofactor can undergo a wide range of transformations, complemented by considerable UbiD-variability. These provide a blueprint for biotechnological applications aimed at producing hydrocarbons or aromatic C-H activation through carboxylation.
Asunto(s)
Carboxiliasas , Dimetilaliltranstransferasa , Flavinas/metabolismo , Carboxiliasas/genética , Carboxiliasas/metabolismo , Mononucleótido de Flavina/química , Oxidación-Reducción , Dimetilaliltranstransferasa/genética , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismoRESUMEN
Prenylation is a post-translational modification (PTM) widely found in primary and secondary metabolism. This modification can enhance the lipophilicity of molecules, enabling them to interact with lipid membranes more effectively. The prenylation of peptides is often carried out by cyanobactin prenyltransferases (PTases) from cyanobacteria. These enzymes are of interest due to their ability to add prenyl groups to unmodified peptides, thus making them more effective therapeutics through the subsequent acquisition of increased membrane permeability and bioavailability. Herein we review the current knowledge of cyanobactin PTases, focusing on their discovery, biochemistry, and bioengineering, and highlight the potential application of them as peptide alkylation biocatalysts to generate peptide therapeutics.
Asunto(s)
Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismo , Péptidos Cíclicos/química , Péptidos , BioingenieríaRESUMEN
Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.
Asunto(s)
Productos Biológicos , Dimetilaliltranstransferasa , Animales , Dimetilaliltranstransferasa/química , Dicetopiperazinas , Ciencia de los Datos , Alcaloides Indólicos/química , Ingeniería de Proteínas , Flavinas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Solventes , Carbono , Especificidad por SustratoRESUMEN
Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.
Asunto(s)
Vías Biosintéticas , Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismo , Arginina/metabolismo , Homoarginina/metabolismo , Guanidina , Péptidos Cíclicos/químicaRESUMEN
The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C-C, C-N, and C-O bond-forming trajectories. DMATS1 from Fusarium fujikuroi catalyzes the reverse N-prenylation of l-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of l-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or l-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C-N and C-O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with l-Trp or l-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of l-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure-function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.
Asunto(s)
Productos Biológicos , Dimetilaliltranstransferasa , Triptófano Sintasa , Catálisis , Dimetilaliltranstransferasa/química , Fusarium , Hemiterpenos , Indoles , Neopreno , Nitrógeno , Compuestos Organofosforados , Prenilación , Especificidad por Sustrato , Triptófano/química , Triptófano Sintasa/metabolismoRESUMEN
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 ß-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.