farnesyl-pyrophosphate and pentalenene

For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used for the transformation of the wild-type strain of X. dendrorhous. The transformations resulted in white colonies, showing a complete shutdown of the carotenoid production. Furthermore, an additional vector was constructed for the insertion of the PSS gene in the rDNA of the yeast. All the mutant strains produce the sesquiterpene pentalenene and show no difference in growth when compared to the wild-type strain. In this report, we demonstrate that X. dendrorhous is a suitable host for the expression of heterologous terpene cyclases and for the production of foreign terpene compounds.

Topics: Basidiomycota; Biocatalysis; Biosynthetic Pathways; Carotenoids; Colony Count, Microbial; Cyclopentanes; Escherichia coli; Gas Chromatography-Mass Spectrometry; Gene Expression; Genes, Bacterial; Intramolecular Lyases; Mutation; Phenotype; Polyisoprenyl Phosphates; Sesquiterpenes; Streptomyces; Transformation, Genetic

In this article, we describe studies, using quantum chemical computations, on possible polycyclization pathways of the farnesyl cation leading to the complex sesquiterpene pentalenene. Two distinct pathways to pentalenene with similar activation barriers are described, each differing from previous mechanistic proposals, and each involving unusual and unexpected intermediates. Direct deprotonation of intermediates on these pathways leads to sesquiterpene byproducts, such as humulene, protoilludene, and asteriscadiene, supporting the notion that a key function of pentalenene synthase, the enzyme that produces pentalenene in Nature, is to regulate the timing and location of proton removal. The implications of the computational results for experimental studies on pentalenene synthase are discussed.

Topics: Cations; Cyclization; Cyclopentanes; Models, Molecular; Polyisoprenyl Phosphates; Quantum Theory; Sesquiterpenes; Thermodynamics

The crystal structure of pentalenene synthase at 2.6 angstrom resolution reveals critical active site features responsible for the cyclization of farnesyl diphosphate into the tricyclic hydrocarbon pentalenene. Metal-triggered substrate ionization initiates catalysis, and the alpha-barrel active site serves as a template to channel and stabilize the conformations of reactive carbocation intermediates through a complex cyclization cascade. The core active site structure of the enzyme may be preserved among the greater family of terpenoid synthases, possibly implying divergence from a common ancestral synthase to satisfy biological requirements for increasingly diverse natural products.

Topics: Alkyl and Aryl Transferases; Binding Sites; Crystallization; Crystallography, X-Ray; Cyclization; Cyclopentanes; Geranyltranstransferase; Intramolecular Lyases; Isomerases; Models, Molecular; Polyisoprenyl Phosphates; Protein Conformation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Sesquiterpenes; Streptomyces; Transferases

Pentalenene synthase, an enzyme which catalyzes the cyclization of farnesyl pyrophosphate to the sesquiterpene hydrocarbon pentalenene, has been partially purified from the supernatant fraction of Streptomyces UC5319 by a combination of anion-exchange, hydroxylapatite, and gel-filtration chromatography. The molecular weight of the partially purified synthase was estimated by gel filtration chromatography to be 57,000 and the cyclase activity was shown to be associated with a major protein band among eight visible by nondenaturing polyacrylamide disc gel electrophoresis. The Km for farnesyl pyrophosphate was 0.77 +/- 0.21 microM and the Vmax for the partially purified synthase was 287 +/- 21 nmol of pentalenene/mg protein per hour. Cyclase activity required the presence of a divalent metal cation. Although either Mg2+ or Mn2+ could be used, Mn2+ was inhibitory at concentrations above 2.5 mM. No other cofactors were required. Whereas neither product, pentalenene nor inorganic pyrophosphate, showed significant inhibition of cyclase activity at concentrations of ca 10 microM, the combination of the two resulted in an approximate sevenfold increase in the apparent Km for farnesyl pyrophosphate, suggesting that both products can bind cooperatively at the active site to inhibit pentalenene synthase competitively.

Topics: Cations, Divalent; Chromatography; Cyclopentanes; Drug Stability; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Intramolecular Lyases; Isomerases; Kinetics; Magnesium; Manganese; Polyisoprenyl Phosphates; Sesquiterpenes; Streptomyces