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.     

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.     

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.     

Molecular characterization of a membrane-bound prenyltransferase specific for isoflavone from Sophora flavescens

Prenylated isoflavones are secondary metabolites that are mainly distributed in legume plants. They often possess divergent biological activities such as anti-bacterial, anti-fungal, and anti-oxidant activities and thus attract much attention in food, medicinal, and agricultural research fields. Prenyltransferase is the key enzyme in the biosynthesis of prenylated flavonoids by catalyzing a rate-limiting step, i.e. the coupling process of two major metabolic pathways, the isoprenoid pathway and shikimate/polyketide pathway. However, so far only two genes have been isolated as prenyltransferases involved in the biosynthesis of prenylated flavonoids, namely naringenin 8-dimethylallyltransferase from Sophora flavescens (SfN8DT-1) specific for some limited flavanones and glycinol 4-dimethylallyltransferase from Glycine max (G4DT), specific for pterocarpan substrate. We have in this study isolated two novel genes coding for membrane-bound flavonoid prenyltransferases from S. flavescens, an isoflavone-specific prenyltransferase (SfG6DT) responsible for the prenylation of the genistein at the 6-position and a chalcone-specific prenyltransferase designated as isoliquiritigenin dimethylallyltransferase (SfiLDT). These prenyltransferases were enzymatically characterized using a yeast expression system. Analysis on the substrate specificity of chimeric enzymes between SfN8DT-1 and SfG6DT suggested that the determinant region for the specificity of the flavonoids was the domain neighboring the fifth transmembrane α-helix of the prenyltransferases.     

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.     

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.     

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.     

HlPT-1, a membrane-bound prenyltransferase responsible for the biosynthesis of bitter acids in hops

Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and β-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg(2+) as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.     

Structure and mechanism of the magnesium-independent aromatic prenyltransferase CloQ from the clorobiocin biosynthetic pathway

CloQ is an aromatic prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans. It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein, we report the crystal structure of CloQ and use it as a framework for interpreting biochemical data from both wild-type and variant proteins. CloQ belongs to the aromatic prenyltransferase family, which is characterized by an unusual core fold comprising five consecutive ααββ elements that form a central 10-stranded anti-parallel β-barrel. The latter delineates a solvent-accessible cavity where substrates bind and catalysis takes place. This cavity has well-defined polar and nonpolar regions, which have distinct roles in substrate binding and facilitate a Friedel-Crafts-type mechanism. We propose that the juxtaposition of five positively charged residues in the polar region circumvents the necessity for a Mg²⁺, which, by contrast, is a strict requirement for the majority of prenyltransferases characterized to date. Our structure of CloQ complexed with 4-hydroxyphenylpyruvate reveals the formation of a covalent link between the substrate and Cys215 to yield a thiohemiketal species. Through site-directed mutagenesis, we show that this link is not essential for enzyme activity in vitro. Furthermore, we demonstrate that CloQ will accept alternative substrates and, therefore, has the capacity to generate a range of prenylated compounds. Since prenylation is thought to enhance the bioactivity of many natural products, CloQ offers considerable promise as a biocatalyst for the chemoenzymatic synthesis of novel compounds with therapeutic potential.     

Evolution of aromatic prenyltransferases in the biosynthesis of indole derivatives

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 different fungi. We have cloned and overexpressed seven of these genes, fgaPT1, fgaPT2, ftmPT1, ftmPT2, 7-dmats, cdpNPT and anaPT in Escherichia coli and Saccharomyces cerevisiae. The overproduced enzymes were characterised biochemically. Three additional indole prenyltransferases, DmaW-Cs, TdiB and MaPT were also identified and characterised in the last years. Sequence analysis and comparison with known aromatic prenyltransferases as well as biochemical investigation revealed that these enzymes belong to a group of aromatic prenyltransferases. The characterised prenyltransferases are soluble proteins, catalyse different prenyl transfer reactions on indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. In addition, indole prenyltransferases carry tryptophan aminopeptidase activity, which strengths their relationship in the evolution. These properties differ clearly from membrane-bound aromatic prenyltransferases from different sources and soluble prenyltransferases from bacteria. All of the indole prenyltransferases accepted only dimethylallyl diphosphate as prenyl donor. On the other hand, they showed broad substrate specificity towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by these enzymes, providing a strategy for convenient production of biologically active substances, e.g. by chemoenzymatic synthesis.     

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.     

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。     

Evolutionary relationships of microbial aromatic prenyltransferases

The linkage of isoprenoid and aromatic moieties, catalyzed by aromatic prenyltransferases (PTases), leads to an impressive diversity of primary and secondary metabolites, including important pharmaceuticals and toxins. A few years ago, a hydroxynaphthalene PTase, NphB, featuring a novel ten-stranded β-barrel fold was identified in Streptomyces sp. strain CL190. This fold, termed the PT-barrel, is formed of five tandem ααββ structural repeats and remained exclusive to the NphB family until its recent discovery in the DMATS family of indole PTases. Members of these two families exist only in fungi and bacteria, and all of them appear to catalyze the prenylation of aromatic substrates involved in secondary metabolism. Sequence comparisons using PSI-BLAST do not yield matches between these two families, suggesting that they may have converged upon the same fold independently. However, we now provide evidence for a common ancestry for the NphB and DMATS families of PTases. We also identify sequence repeats that coincide with the structural repeats in proteins belonging to these two families. Therefore we propose that the PT-barrel arose by amplification of an ancestral ααββ module. In view of their homology and their similarities in structure and function, we propose to group the NphB and DMATS families together into a single superfamily, the PT-barrel superfamily.     

Towards complete sets of farnesylated and geranylgeranylated proteins

Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.     

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.     

Structural studies on human 2-oxoglutarate dependent oxygenases

2-Oxoglutarate and ferrous iron-dependent oxygenases have emerged as an important family of human enzymes that catalyse hydroxylations and related demethylation reactions. Their substrates in humans include proteins, nucleic acids, lipids and small molecules. They play roles in collagen biosynthesis, hypoxic sensing, regulation of gene expression and lipid biosynthesis/metabolism. Structural analyses, principally employing crystallography, have revealed that all of these oxygenases possess a double-stranded β-helix core fold that supports a highly conserved triad of iron binding residues and a less well conserved 2-oxoglutarate co-substrate binding site. The 2-oxoglutarate binds to the iron in a bidentate manner via its 1-carboxylate and 2-oxo groups. The primary substrate binding elements are more variable and can involve mobile elements.     

Structure, mechanism and function of prenyltransferases.

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.     

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.