Publications

• Biochemical and genomic characterization of terpene synthases in Magnolia grandiflora.

  Lee S., Chappell J.

  Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (T ... >> More

  Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated, and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted farnesyl diphosphate (C(15)) predominantly to beta-cubebene, while Mg17 converted geranyl diphosphate (C(5)) to alpha-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all three genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 messenger RNAs in stamens. A putative amino-terminal signal peptide of Mg17 targeted the reporter green fluorescent protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, the intron-exon organizations for the three Magnolia TPS genes were different from one another and from other well-characterized TPS gene sets. The Mg17 gene consists of six introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only four introns, and Mg25 has only a single intron located near the 5' terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene. << Less

  Plant Physiol. 147:1017-1033(2008) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Sesquiterpene synthases Cop4 and Cop6 from Coprinus cinereus: catalytic promiscuity and cyclization of farnesyl pyrophosphate geometric isomers.

  Lopez-Gallego F., Agger S.A., Abate-Pella D., Distefano M.D., Schmidt-Dannert C.

  Sesquiterpene synthases catalyze with different catalytic fidelity the cyclization of farnesyl pyrophosphate (FPP) into hundreds of known compounds with diverse structures and stereochemistries. Two sesquiterpene synthases, Cop4 and Cop6, were previously isolated from Coprinus cinereus as part of ... >> More

  Sesquiterpene synthases catalyze with different catalytic fidelity the cyclization of farnesyl pyrophosphate (FPP) into hundreds of known compounds with diverse structures and stereochemistries. Two sesquiterpene synthases, Cop4 and Cop6, were previously isolated from Coprinus cinereus as part of a fungal genome survey. This study investigates the reaction mechanism and catalytic fidelity of the two enzymes. Cyclization of all-trans-FPP ((E,E)-FPP) was compared to the cyclization of the cis-trans isomer of FPP ((Z,E)-FPP) as a surrogate for the secondary cisoid neryl cation intermediate generated by sesquiterpene synthases, which are capable of isomerizing the C2--C3 pi bond of all-trans-FPP. Cop6 is a "high-fidelity" alpha-cuprenene synthase that retains its fidelity under various conditions tested. Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)-FPP into multiple products, including (-)-germacrene D and cubebol. Changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme. Cyclization of (Z,E)-FPP by Cop4 and Cop6 yields products that are very different from those obtained with (E,E)-FPP. Conversion of (E,E)-FPP proceeds via a (6R)-beta-bisabolyl carbocation in the case of Cop6 and an (E,E)-germacradienyl carbocation in the case of Cop4. However, (Z,E)-FPP is cyclized via a (6S)-beta-bisabolene carbocation by both enzymes. Structural modeling suggests that differences in the active site and the loop that covers the active site of the two enzymes might explain their different catalytic fidelities. << Less

  ChemBioChem 11:1093-1106(2010) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus.

  Agger S., Lopez-Gallego F., Schmidt-Dannert C.

  Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in pl ... >> More

  Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an alpha-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes delta-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of alpha-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated alpha-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species. << Less

  Mol. Microbiol. 72:1181-1195(2009) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Selectivity of fungal sesquiterpene synthases: role of the active site's H-1 alpha loop in catalysis.

  Lopez-Gallego F., Wawrzyn G.T., Schmidt-Dannert C.

  Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterp ... >> More

  Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases. << Less

  Appl. Environ. Microbiol. 76:7723-7733(2010) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.