Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.     

Prenyl transfer to aromatic substrates in the biosynthesis of aminocoumarins,meroterpenoids and phenazines: The ABBA prenyltransferase family

Aromatic prenyltransferases transfer prenyl moieties onto aromatic acceptor molecules, catalyzing an electrophilic substitution of the aromatic ring under formation of carbon–carbon bonds. They give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. This review describes a recently discovered family of aromatic prenyltransferases. The structure of these enyzmes shows a type of β/α fold with antiparallel β strands. Due to the α-β-β-α architecture of this fold, this group of enzymes was designated as ABBA prenyltransferases. They lack the (N/D)DxxD motif which is characteristic for many other prenyltransferases.At present, 14 genes with sequence similarity to ABBA prenyltransferases can be identified in the database. A phylogenetic analysis of these genes separates them into two clades. One of them comprises the 4-hydroxyphenylpyruvate 3-dimethylallyltransferases CloQ and NovQ involved in aminocoumarin antibiotic biosynthesis in Streptomyces strains, as well as four genes of unknown function from fungal genomes. The other clade comprises genes involved in the biosynthesis of prenylated naphthoquinones and prenylated phenazines in different streptomycetes. ABBA prenyltransferases are soluble biocatalysts which can easily be obtained as homogeneous proteins in significant amounts. Their substrates are accommodated in a surprisingly spacious central cavity which explains their promiscuity for different aromatic substrates. Therefore, the enzymes of this family represent attractive tools for the chemoenzymatic synthesis of bioactive molecules.     

芳香族异戊烯转移酶的研究进展

异戊烯基转移酶(prenyltransferase)催化异戊烯基转移至异戊烯单元、芳香环或蛋白质上。芳香族异戊烯基转移酶将异戊烯单元融入含有芳环的化合物, 从而形成具有重要生物学功能的各类活性分子, 如泛醌、质体醌、维生素E、异戊烯黄酮类以及真菌代谢物等。该文综述了近年来植物和真菌芳香族异戊烯转移酶的分子生物学研究进展, 包括膜结合的参与质体醌生物合成的homogentisate solanesyltransferase、参与维生素E生物合成的homogentisate phytyltransferase、类黄酮异戊烯转移酶(flavonoid prenyltransferase)和可溶性的真菌吲哚异戊烯转移酶等。     

Prenyltransferases as key enzymes in primary and secondary metabolism

Attachment of isoprene units to various acceptors by prenylation plays an important role in primary and secondary metabolism of living organisms. Protein prenylation belongs to posttranslational modification and is involved in cellular regulation process. Prenylated secondary metabolites usually demonstrate promising biological and pharmacological activities. Prenyl transfer reactions catalyzed by prenyltransferases represent the key steps in the biosynthesis and contribute significantly to the structural and biological diversity of these compounds. In the last decade, remarkable progress has been achieved in the biochemical, molecular, and structural biological investigations of prenyltransferases, especially on those of the members of the dimethylallyltryptophan synthase (DMATS) superfamily. Until now, more than 40 of such soluble enzymes are identified and characterized biochemically. They catalyze usually regioselective and stereoselective prenylations of a series of aromatic substances including tryptophan, tryptophan-containing peptides, and other indole derivatives as well as tyrosine or even nitrogen-free substrates. Crystal structures of a number of prenyltransferases have been solved in the past 10 years and provide a solid basis for understanding the mechanism of prenyl transfer reactions.     

芳香族异戊烯转移酶的研究进展

异戊烯基转移酶（prenyltransferase）催化异戊烯基转移至异戊烯单元、芳香环或蛋白质上。芳香族异戊烯基转移酶将异戊烯单元融入含有芳环的化合物,从而形成具有重要生物学功能的各类活性分子,如泛醌、质体醌、维生素E、异戊烯黄酮类以及真菌代谢物等。该文综述了近年来植物和真菌芳香族异戊烯转移酶的分子生物学研究进展,包括膜结合的参与质体醌生物合成的homogentisate solanesyltransferase、参与维生素E生物合成的homogentisate phytyltransferase、类黄酮异戊烯转移酶（flavonoid prenyltransferase）和可溶性的真菌吲哚异戊烯转移酶等。     

Molecular and structural basis of metabolic diversity mediated by prenyldiphosphate converting enzymes

General thermodynamic calculations using the semiempiric PM3 method have led to the conclusion that prenyldiphosphate converting enzymes require at least one divalent metal cation for the activation and cleavage of the diphosphate–prenyl ester bond, or they must provide structural elements for the efficient stabilization of the intermediate prenyl cation. The most important common structural features, which guide the product specificity in both terpene synthases and aromatic prenyl transferases are aromatic amino acid side chains, which stabilize prenyl cations by cation–π interactions. In the case of aromatic prenyl transferases, a proton abstraction from the phenolic hydroxyl group of the second substrate will enhance the electron density in the phenolic ortho-position at which initial prenylation of the aromatic compound usually occurs.A model of the structure of the integral transmembrane-bound aromatic prenyl transferase UbiA was developed, which currently represents the first structural insight into this group of prenylating enzymes with a fold different from most other aromatic prenyl transferases. Based on this model, the structure–activity relationships and mechanistic aspects of related proteins, for example those of Lithospermum erythrorhizon or the enzyme AuaA from Stigmatella aurantiaca involved in the aurachin biosynthesis, were elucidated. The high similarity of this group of aromatic prenyltransferases to 5-epi-aristolochene synthase is an indication of an evolutionary relationship with terpene synthases (cyclases). This is further supported by the conserved DxxxD motif found in both protein families. In contrast, there is no such relationship to the aromatic prenyl transferases with an ABBA-fold, such as NphB, or to any other known family of prenyl converting enzymes. Therefore, it is possible that these two groups might have different evolutionary ancestors.     

Prenylation of aromatic compounds,a key diversification of plant secondary metabolites

Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. A prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (?)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.     

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.     

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.     

Structure of a Membrane-Embedded Prenyltransferase Homologous to UBIAD1

Membrane-embedded prenyltransferases from the UbiA family catalyze the Mg²⁺-dependent transfer of a hydrophobic polyprenyl chain onto a variety of acceptor molecules and are involved in the synthesis of molecules that mediate electron transport, including Vitamin K and Coenzyme Q. In humans, missense mutations to the protein UbiA prenyltransferase domain-containing 1 (UBIAD1) are responsible for Schnyder crystalline corneal dystrophy, which is a genetic disease that causes blindness. Mechanistic understanding of this family of enzymes has been hampered by a lack of three-dimensional structures. We have solved structures of a UBIAD1 homolog from Archaeoglobus fulgidus, AfUbiA, in an unliganded form and bound to Mg²⁺ and two different isoprenyl diphosphates. Functional assays on MenA, a UbiA family member from E. coli, verified the importance of residues involved in Mg²⁺ and substrate binding. The structural and functional studies led us to propose a mechanism for the prenyl transfer reaction. Disease-causing mutations in UBIAD1 are clustered around the active site in AfUbiA, suggesting the mechanism of catalysis is conserved between the two homologs.     

Biochemical characterization of indole prenyltransferases: filling the last gap of prenylation positions by a 5-dimethylallyltryptophan synthase from Aspergillus clavatus

The putative prenyltransferase gene ACLA_031240 belonging to the dimethylallyltryptophan synthase superfamily was identified in the genome sequence of Aspergillus clavatus and overexpressed in Escherichia coli. The soluble His-tagged protein EAW08391 was purified to near homogeneity and used for biochemical investigation with diverse aromatic substrates in the presence of different prenyl diphosphates. It has shown that in the presence of dimethylallyl diphosphate (DMAPP), the recombinant enzyme accepted very well simple indole derivatives with L-tryptophan as the best substrate. Product formation was also observed for tryptophan-containing cyclic dipeptides but with much lower conversion yields. In contrast, no product formation was detected in the reaction mixtures of L-tryptophan with geranyl or farnesyl diphosphate. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific regular prenylation at C-5 of the indole nucleus of the simple indole derivatives. EAW08391 was therefore termed 5-dimethylallyltryptophan synthase, and it filled the last gap in the toolbox of indole prenyltransferases regarding their prenylation positions. K(m) values of 5-dimethylallyltryptophan synthase were determined for L-tryptophan and DMAPP at 34 and 76 μM, respectively. Average turnover number (k(cat)) at 1.1 s(-1) was calculated from kinetic data of L-tryptophan and DMAPP. Catalytic efficiencies of 5-dimethylallyltryptophan synthase for L-tryptophan at 25,588 s(-1)·M(-1) and for other 11 simple indole derivatives up to 1538 s(-1)·M(-1) provided evidence for its potential usage as a catalyst for chemoenzymatic synthesis.     

Aromatic Prenylation in Phenazine Biosynthesis: DIHYDROPHENAZINE-1-CARBOXYLATE DIMETHYLALLYLTRANSFERASE FROM STREPTOMYCES ANULATUS*S?

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The K_m value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. K_cat was calculated as 0.435 s^-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives rise to an astounding diversity of secondary metabolites in bacteria, fungi, and plants, including many compounds that are important in pharmacotherapy. However, surprisingly little biochemical and genetic data are available on the enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new family of aromatic prenyltransferases was discovered in streptomycetes (1), Gram-positive soil bacteria that are prolific producers of antibiotics and other biologically active compounds (2). The members of this enzyme family show a new type of protein fold with a unique α-β-β-α architecture (3) and were therefore termed ABBA prenyltransferases (1). Only 13 members of this family can be identified by sequence similarity searches in the data base at present, and only four of them have been investigated biochemically (3–6). Up to now, only phenolic compounds have been identified as aromatic substrates of ABBA prenyltransferases. We now report the discovery of a new member of the ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate (dihydro-PCA).² Streptomyces strains produce many of prenylated phenazines as natural products. For the first time, the present paper reports the identification of a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces several prenylated phenazines, among them endophenazine A and B (Fig. 1A) (7). We wanted to investigate which type of prenyltransferase might catalyze the prenylation reaction in endophenazine biosynthesis. In streptomycetes and other microorganisms, genes involved in the biosynthesis of a secondary metabolite are nearly always clustered in a contiguous DNA region. Therefore, the prenyltransferase of endophenazine biosynthesis was expected to be localized in the vicinity of the genes for the biosynthesis of the phenazine core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663. B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG is responsible for the biosynthesis of PCA (8). The enzyme PhzC catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate (i.e. the first step of the shikimate pathway), and further enzymes of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze the conversion of chorismate to 2-amino-2-deoxyisochorismate and the subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively. These reactions are well established biochemically. Fewer data are available about the following steps (i.e. dimerization of 2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a decarboxylation, ultimately leading to PCA via several instable intermediates). From Pseudomonas, experimental data on the role of PhzF and PhzA/B have been published (8, 9), whereas the role of PhzG is yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis described so far from streptomycetes (10) was found not to contain a phzF orthologue, raising the question of whether there may be differences in the biosynthesis of phenazines between Pseudomonas and Streptomyces.Screening of a genomic library of the endophenazine producer strain S. anulatus now allowed the identification of the first complete gene cluster of a prenylated phenazine, including the structural gene of dihydro-PCA dimethylallyltransferase.     

Structures,mechanisms and inhibitors of undecaprenyl diphosphate synthase: A cis-prenyltransferase for bacterial peptidoglycan biosynthesis

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.     

Diversity of ABBA Prenyltransferases in Marine Streptomyces sp. CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 –CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.     

Characterisation of novel functionality within the Blastocystis tryptophanase gene

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.     

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.     

A New Group of Aromatic Prenyltransferases in Fungi,Catalyzing a 2,7-Dihydroxynaphthalene 3-Dimethylallyl-transferase Reaction

Five fungal genomes from the Ascomycota (sac fungi) were found to contain a gene with sequence similarity to a recently discovered small group of bacterial prenyltransferases that catalyze the C-prenylation of aromatic substrates in secondary metabolism. The genes from Aspergillus terreus NIH2624, Botryotinia fuckeliana B05.10 and Sclerotinia sclerotiorum 1980 were expressed in Escherichia coli, and the resulting His₈-tagged proteins were purified and investigated biochemically. Their substrate specificity was found to be different from that of any other prenyltransferase investigated previously. Using 2,7-dihydroxynaphthalene (2,7-DHN) and dimethylallyl diphosphate as substrates, they catalyzed a regiospecific Friedel-Crafts alkylation of 2,7-DHN at position 3. Using the enzyme of A. terreus, the K_m values for 2,7-DHN and dimethylallyl diphosphate were determined as 324 ± 25 μm and 325 ± 35 μm, respectively, and k_cat as 0.026 ± 0.001 s⁻¹. A significantly lower level of prenylation activity was found using dihydrophenazine-1-carboxylic acid as aromatic substrate, and only traces of products were detected with aspulvinone E, flaviolin, or 4-hydroxybenzoic acid. No product was formed with l-tryptophan, l-tyrosine, or 4-hydroxyphenylpyruvate. The genes for these fungal prenyltransferases are not located within recognizable secondary metabolic gene clusters. Their physiological function is yet unknown.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Unusual N-Prenylation in Diazepinomicin Biosynthesis: The Farnesylation of a Benzodiazepine Substrate Is Catalyzed by a New Member of the ABBA Prenyltransferase Superfamily

The bacterium Micromonospora sp. RV115, isolated from a marine sponge, produces the unusual metabolite diazepinomicin, a prenylated benzodiazepine derivative. We have cloned the prenyltransferase gene dzmP from this organism, expressed it in Escherichia coli, and the resulting His₈-tagged protein was purified and investigated biochemically. It was found to catalyze the farnesylation of the amide nitrogen of dibenzodiazepinone. DzmP belongs to the ABBA prenyltransferases and is the first member of this superfamily which utilizes farnesyl diphosphate as genuine substrate. All previously discovered members utilize either dimethylallyl diphosphate (C₅) or geranyl diphosphate (C₁₀). Another putative diazepinomicin biosynthetic gene cluster was identified in the genome of Streptomyces griseoflavus Tü4000, suggesting that the formation of diazepinomicin is not restricted to the genus Micromonospora. The gene cluster contains a gene ssrg_00986 with 61.4% identity (amino acid level) to dzmP. The gene was expressed in E. coli, and the purified protein showed similar catalytic properties as DzmP. Both enzymes also accepted other phenolic or phenazine substrates. ABBA prenyltransferases are useful tools for chemoenzymatic synthesis, due to their nature as soluble, stable biocatalysts. The discovery of DzmP and Ssrg_00986 extends the isoprenoid substrate range of this superfamily. The observed prenylation of an amide nitrogen is an unusual biochemical reaction.