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
Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.The naturally occurring phenazines include more than 50 nitrogen-containing heterocyclic pigments of bacterial origin (36). They have characteristic absorption spectra with two peaks in the UV range and at least one peak in the visible range that determines their colors (42). Almost all phenazines are broadly inhibitory to the growth of bacteria and fungi due to their ability to undergo cellular redox cycling in the presence of oxygen and reducing agents (including NADH and NADPH) and cause the accumulation of toxic superoxide and hydrogen peroxide (42). Phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine-1-carboxylic acid, and phenazine-1-carboxamide (PCN) produced in the rhizosphere by Pseudomonas fluorescens and Pseudomonas chlororaphis inhibit soilborne phytopathogenic fungi (13, 62) and contribute to the natural suppression of Fusarium wilt disease in certain soils in France (45). Phenazines produced by Pantoea agglomerans on apple flowers contribute to suppression of phytopathogenic Erwinia amylovora, which causes fire blight disease (22). Production of pyocyanin (PYO) by Pseudomonas aeruginosa is required for generation of disease symptoms in plants and killing of the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster (40, 54), and it is also critical for lung infection by P. aeruginosa in mice (35). In contrast, some phenazines produced by Streptomyces spp. are not cytotoxic in eukaryotes and have promise as anticancer or anti-infective drugs (36).In addition to the effects of phenazines on other organisms, recent studies have indicated that these compounds directly activate certain transcription factors and act as intercellular signals in P. aeru...
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...
Aromatic prenyltransferases (PTases) catalyze the transfer of a C5 (dimethylallyl), C10 (geranyl) or C15 (farnesyl) prenyl group derived from the corresponding isoprenyl diphosphate metabolites onto a variety of electron-rich aromatic acceptors. Prenyl groups appear in a wide variety of bioactive natural products of microbial and plant origin, including amino acids, stilbenes, alkaloids, polyketides and phenylpropanoids such as flavonoids, creating natural product hybrids with altered or enhanced bioactivities. Prenylation of flavonoids enhances some of the desirable pharmacological properties of these plant compounds [1], as demonstrated for apigenin and liquiritigenin [2]. Prenylation appears in many cases to provide a higher level of bioactivity compared to the non-prenylated precursor, often by increasing affinity for biological membranes and interactions with cellular targets [3]. With the recent identification of these enzymes there is increased interest in the role of these regiospecific catalysts in expanding the diversity and bioactivities of several important classes of natural products in vivo and in vitro.One way in which PTases can be categorized depends on whether they catalyze the synthesis of isoprenyl diphosphates, the prenylation of a protein or the prenylation of an aromatic substrate. Isoprenyl diphosphate synthases catalyze the chain elongation of an allylic isoprenyl diphosphate substrate by reaction with isopentenyl diphosphate [4]. Protein PTases transfer a geranyl-geranyl or farnesyl group to the Cys residue on a CaaX motif at the C-terminus of several proteins to facilitate membrane anchoring in eukaryotes and possibly archaea [5]. Small-molecule aromatic PTases constituting the third category can be subdivided into membrane-associated and functionally soluble PTases. Membrane-associated PTases contain a characteristic (N/D)DXXD Mg 2+ -diphosphate binding motif which is also found in the isoprenyl diphosphate synthases and are involved e.g. in the biosynthesis of ubiquinones and © Birkhäuser Verlag, Basel, 2008 Richard@salk.edu. [6], in the biosynthesis of membrane lipids in archaea [7] and in the formation of plant secondary metabolites [8].The functionally soluble PTases do not possess an obvious Mg 2+ -diphosphate binding motif. Moreover, most of the currently known soluble aromatic PTases segregate into two subgroups based on their primary sequence similarity and on the small-molecule substrates they accept for prenylation. The first subgroup commonly prenylates indole-containing ring systems. Examples of these PTases include the fungal enzymes FgaPT1 and FgaPT2 involved in the biosynthesis of fumigaclavin C [9], as well as the newly discovered N-reverse PTase CdpNPT [10] and the 7-dimethylallyl-tryptophan synthase Afu3 g12930 [11]. LtxC, which is involved in the biosynthesis of lyngbyatoxins obtained from the cyanobacteria Lyngbya majuscula [12], also prenylates an indole-containing ring system but shares no sequence similaritywith the fungal enzymes named above.The second subg...
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