Prenylated indole alkaloids are hybrid natural products derived from prenyl diphosphates and tryptophan or its precursors and widely distributed in filamentous fungi, especially in the genera Penicillium and Aspergillus of ascomycota. These compounds represent a group of natural products with diverse chemical structures and biological activities. Significant progress on their biosynthesis has been achieved in recent years by identification of biosynthetic gene clusters from genome sequences and by molecular biological and biochemical investigations. In addition, a series of prenylated indole derivatives have been produced by chemoenzymatic synthesis using overproduced and purified enzymes.

Ergot alkaloids are toxins and important pharmaceuticals that are produced biotechnologically on an industrial scale. The first committed step of ergot alkaloid biosynthesis is catalyzed by dimethylallyl tryptophan synthase (DMATS; EC 2.5.1.34). Orthologs of DMATS are found in many fungal genomes. We report here the x-ray structure of DMATS, determined at a resolution of 1.76 Å. A complex of DMATS from Aspergillus fumigatus with its aromatic substrate L-tryptophan and with an analogue of its isoprenoid substrate dimethylallyl diphosphate reveals the structural basis of this enzyme-catalyzed Friedel-Crafts reaction, which shows strict regiospecificity for position 4 of the indole nucleus of tryptophan as well as unusual independence of the presence of Mg 2؉ ions. The 3D structure of DMATS belongs to a rare ␤/␣ barrel fold, called prenyltransferase barrel, that was recently discovered in a small group of bacterial enzymes with no sequence similarity to DMATS. These bacterial enzymes catalyze the prenylation of aromatic substrates in the biosynthesis of secondary metabolites (i.e., a reaction similar to that of DMATS).EC 2.5.1.34 ͉ ergot alkaloids ͉ PT barrel ͉ ABBA prenyltransferase

A putative prenyltransferase gene, ftmPT1, was identified in the genome sequence of Aspergillus fumigatus. ftmPT1 was cloned and expressed in Escherichia coli, and the protein FtmPT1 was purified to near homogeneity and characterized biochemically. This enzyme was found to catalyse the prenylation of cyclo-L-Trp-L-Pro (brevianamide F) at the C-2 position of the indole nucleus. FtmPT1 is a soluble monomeric protein, which does not contain the usual prenyl diphosphate binding site (N/D)DXXD found in most prenyltransferases, and which does not require divalent metal ions for its enzymic activity. K m values for brevianamide F and dimethylallyl diphosphate were determined as 55 and 74 mM, respectively. The turnover number was 5?57 s "1 . FtmPT1 showed a high substrate specificity towards dimethylallyl diphosphate, but accepted different tryptophan-containing cyclic dipeptides. Together with dimethylallyltryptophan synthase of ergot alkaloid biosynthesis, FtmPT1 belongs to a new group of prenyltransferases with aromatic substrates. INTRODUCTIONTrans-prenyltransferases (Bohlmann et al., 1998;Liang et al., 2002;Sacchettini & Poulter, 1997) are involved in the biosynthesis of terpenoids from C-5 units. These enzymes usually contain one or more prenyl diphosphate binding motifs, (N/D)DXXD, in their sequences (Bohlmann et al., 1998;Liang et al., 2002;Sacchettini & Poulter, 1997). Their enzymic reaction depends strictly on the presence of metal ions such as Mg 2+ or Mn 2+ (Bohlmann et al., 1998;Liang et al., 2002). A special group of prenyltransferases catalyses the attachment of a prenyl moiety to an aromatic nucleus. These enzymes show similar sequence motifs and metal ion requirements to those of the trans-prenyltransferases. Examples of this group are the prenyltransferases involved in the biosynthesis of the primary metabolites ubiquinone (Turunen et al., 2004), menaquinone (Suvarna et al., 1998), tocopherol (Schledz et al., 2001) and plastoquinone (Collakova & DellaPenna, 2001), and the prenyltransferase involved in the formation of the plant secondary metabolite shikonin (Yazaki et al., 2002). All these enzymes are membrane-bound proteins.In contrast, the dimethylallyltryptophan synthase (DMATS) that catalyses the prenylation of tryptophan at position C-4 of the indole nucleus during ergot alkaloid biosynthesis in the fungus Claviceps has been found to be a soluble protein and is active in a metal-free buffer containing EDTA (Cress et al., 1981;Lee et al., 1976;Tsai et al., 1995; Tudzynski et al., 1999). Recently, two further soluble prenyltransferases with aromatic substrates, which are active in the absence of metal ions and contain no (N/D)DXXD motifs, CloQ involved in the biosynthesis of clorobiocin from Streptomyces roseochromogenes (Pojer et al., 2003) and LtxC involved in the biosynthesis of lyngbyatoxins from Lyngbya majuscula (Edwards & Gerwick 2004), have been identified. Very recently, we identified an orthologue of DMATS, FgaPT2, in the genome sequence of Aspergillus fumigatus (Unsöld & Li, 2005). Inte...

A putative dimethylallyltryptophan synthase gene, fgaPT2, was identified in the genome sequence of Aspergillus fumigatus. fgaPT2 was cloned and overexpressed in Saccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert L-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. K m values for L-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 mM, respectively. Metal ions, such as Mg 2+ and Ca 2+ , enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

Lipid rafts are involved in the life cycle of many viruses. In this study, we showed that lipid rafts also play an important role in the life cycle of severe acute respiratory syndrome (SARS)-coronavirus (CoV). Cholesterol depletion by pretreatment of Vero E6 cells with methyl-beta-cyclodextrin (MbetaCD) inhibited the production of SARS-CoV particles released from the infected cells. This inhibition was prevented by addition of cholesterol to the culture medium, indicating that the reduction of virus particle release was caused by the loss of cholesterol in the cell membrane. In contrast, cholesterol depletion at the post-entry stage (3h post-infection) caused only a limited effect on virus particle release. Northern blot analysis revealed that the levels of viral mRNAs were significantly affected by pretreatment with MbetaCD, but not by treatment at 3h post-infection. Interestingly, no apparent evidence for colocalization of angiotensin converting enzyme 2 with lipid rafts in the membrane of Vero E6 cells was obtained. These results suggest that lipid rafts could contribute to SARS-CoV infection in the early replication process in Vero E6 cells.

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. They have been identified in two orders of fungi and three families of higher plants. The most important producers are fungi of the genera Claviceps, Penicillium and Aspergillus (all belonging to the Ascomycota). Chemically, ergot alkaloids are characterised by the presence of a tetracyclic ergoline ring, and can be divided into three classes according to their structural features, i.e. amide- or peptide-like amide derivatives of D-lysergic acid and the clavine alkaloids. Significant progress has been achieved on the molecular biological and biochemical investigations of ergot alkaloid biosynthesis in the last decade. By gene cloning and genome mining, gene clusters for ergot alkaloid biosynthesis have been identified in at least 8 different ascomycete species. Functions of most structure genes have been assigned to reaction steps in the biosynthesis of ergot alkaloids by gene inactivation experiments or biochemical characterisation of the overproduced proteins.

The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Doublestranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB r , and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.Novobiocin is produced by Streptomyces spheroides and Streptomyces niveus and belongs to the aminocoumarin antibiotics. Bacterial DNA gyrase represents the target of these coumarins (41), and novobiocin inhibits this enzyme by interaction with the N-terminal 24-kDa subdomain of the gyrB subunit (27). In addition to its antibacterial action, novobiocin shows synergistic effects with antitumor drugs such as etoposide or teniposide (37,49).Little is known about the biosynthesis of novobiocin. Structurally, it is composed of three moieties, a noviose sugar (ring C), a substituted coumarin (ring B), and a prenylated 4-hydroxybenzoic acid (ring A), and these rings are linked by glycosidic and amide bonds (Fig. 1). Radioactive feeding experiments in the 1960s and 1970s showed that noviose is directly derived from D-glucose, whereas tyrosine serves as a precursor of ring A and ring B (3, 6, 31). This was recently confirmed by a feeding experiment with [1-13 C]glucose (33) which also showed that the dimethylallyl moiety of novobiocin was formed through the nonmevalonate pathway.Molecular biological studies have been restricted to the investigation of novobiocin resistance genes (43, 52), especially gyrB r (61, 62), and the production of novobiocin-deficient mutants (19). Discovery of the genetic basis of the biosynthesis of aminocoumarin antibiotics could provide a useful tool for drug development. "Combinatorial biosynthesis," the interchange of genes involved in antibiotic biosynthesis between different microorganisms or the creation of hybrid genes and, consequently, proteins with new enzymatic properties, allows the production of modified or even novel antibiotics (23). In the past, much effort has been undertaken in the manipulation of the biosynthesis of polyketide antibiotics (25,42,56), and recently, progress has also been made in the construction of hybrid peptide synthetase genes (55, 59). The discovery of gene clusters for other types of secondary metabolites can offer additional possibilities for combinatorial biosynthesis.Here we report on the identification of the novobiocin biosynthetic gene cluster from S. spheroides NCIB 11891. The gene...

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