Publications

• Monoterpene synthases from gymnosperms and angiosperms: stereospecificity and inactivation by cysteinyl- and arginyl-directed modifying reagents.

  Savage T.J., Ichii H., Hume S.D., Little D.B., Croteau R.

  To further define specific structural and mechanistic differences among monoterpene synthases from divergent plant sources, the stereospecificity of the enzyme-catalyzed isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which hav ... >> More

  To further define specific structural and mechanistic differences among monoterpene synthases from divergent plant sources, the stereospecificity of the enzyme-catalyzed isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which have been well established for monoterpene synthases from herbaceous angiosperms) were examined for monoterpene synthases from a conifer, lodgepole pine (Pinus contorta). The chiral monoterpenes isolated from lodgepole pine oleoresin and the major chiral products from cell-free assays of each of the four lodgepole pine monoterpene synthases belonged to the stereochemical family related by the biosynthetic intermediacy of 3S-linalyl pyrophosphate. Furthermore, both the putative intermediate, 3S-linalyl pyrophosphate, and the natural substrate, geranyl pyrophosphate, were enzymatically converted to the same monoterpene enantiomers. Thus, like monoterpene synthases from herbaceous angiosperms, monoterpene synthases from lodgepole pine appear to catalyze both the stereospecific isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound intermediate to multiple, stereochemically related monoterpene olefin isomers. The susceptibility of monoterpene synthases to inactivation by cysteinyl- and arginyl-directed chemical modification reagents was also examined to identify specific structural differences between enzymes from conifers and angiosperms. Like monoterpene synthases from peppermint (Mentha x piperita) and culinary sage (Salvia officinalis), monoterpene synthases from lodgepole pine were inactivated by thiol-directed reagents; however, unlike monoterpene synthases from these herbaceous angiosperms, monoterpene synthases from lodgepole pine were not protected against inactivation by coincubation with substrate and metal ion cofactor. Lodgepole pine monoterpene synthases were also inactivated by the arginyl-directed reagent phenylglyoxal, and coincubation with substrate and cofactor, to effect active-site protection, reduced the rate of inactivation 10-fold. (+)-Pinene synthase and (-)-pinene synthase from sage were also inactivated by phenylglyoxal, but no protection was afforded by coincubation with substrate and cofactor. Thus, monoterpene synthases of conifers appear to have catalytically important arginyl residues specifically located at or near the active site and have at least some catalytically important thiol residues at a non-substrate-protectable region of the enzyme, in contrast to monoterpene synthases from angiosperms which appear to have catalytically important cysteinyl residues at the active site and have catalytically important arginyl residues located at a non-substrate-protectable region of the enzyme. << Less

  Arch Biochem Biophys 320:257-265(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biosynthesis of monoterpenes: regio- and stereochemistry of (+)-3-carene biosynthesis.

  Savage T.J., Croteau R.

  Incubation of [1-3H1]geraniol with stem disks of Douglas fir (Pseudotsuga menziesii) and incubation of [1-3H1]geranyl pyrophosphate with both a soluble enzyme extract from Douglas fir and a partially purified preparation of (+)-3-carene synthase from lodgepole pine (Pinus contorta) resulted in the ... >> More

  Incubation of [1-3H1]geraniol with stem disks of Douglas fir (Pseudotsuga menziesii) and incubation of [1-3H1]geranyl pyrophosphate with both a soluble enzyme extract from Douglas fir and a partially purified preparation of (+)-3-carene synthase from lodgepole pine (Pinus contorta) resulted in the production of (+)-3-[3H] carene. Subsequent conversion of the product to car-3-en-5-one and to 4-isocaranone followed by base-catalyzed exchange of the alpha-hydrogens established that the 3H located at C1 in the geranyl substrate resided at C5 of (+)-3-carene. Incubation of the (+)-3-carene synthase preparation with (S)-[5-3H1, 4-14C]geranyl pyrophosphate resulted in the production of (+)-3-carene without loss of tritium, indicating that the 5-proR hydrogen is eliminated during cyclopropyl ring closure. Analysis of the conformational requirements for this 1,3 elimination involving the 5-proR hydrogen suggested that cyclopropyl ring formation occurs via a (4S)-alpha-terpinyl cation derived from the anti-endo cyclization of a (3S)-linalyl pyrophosphate intermediate. Kinetic analyses of the conversion of (1Z,3R)-[1-3H1]linalyl pyrophosphate, (1Z, 3S)-[1-3H1]linalyl pyrophosphate and [1-3H1]geranyl pyrophosphate by (+)-3-carene synthase revealed that the velocity of the reaction with the (3S)-linalyl enantiomer was 25-fold greater than the velocity with the (3R)-enantiomer and twice that of the natural substrate, geranyl pyrophosphate, thereby confirming this stereochemical prediction and also indicating that the cyclization of the linalyl intermediate is faster than the coupled isomerization and cyclization of the geranyl substrate. From these results, a model that details the regio- and stereochemistry of the enzymatic conversion of geranyl pyrophosphate to (+)-3-carene is proposed. << Less

  Arch Biochem Biophys 305:581-587(1993) [PubMed] [EuropePMC]

• An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil.

  Hall D.E., Robert J.A., Keeling C.I., Domanski D., Quesada A.L., Jancsik S., Kuzyk M.A., Hamberger B., Borchers C.H., Bohlmann J.

  Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the ... >> More

  Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies. << Less

  Plant J. 65:936-948(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Monoterpene synthases of Pinus contorta and related conifers. A new class of terpenoid cyclase.

  Savage T.J., Hatch M.W., Croteau R.

  A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand ... >> More

  A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand fir (Abies grandis) and other higher plants in molecular mass (67 +/-2 kDa as estimated by size-exclusion chromatography), Km for geranyl pyrophosphate (7.8 +/- 1.9 microM), and isoelectric point (4.75 +/-0.2 as determined by isoelectric focusing), but the cyclases from both lodgepole pine and grand fir are unlike previously characterized terpenoid cyclases from angiosperms and fungi, in that they have an alkaline pH optimum (pH 7.8), are activated by K+, Rb+, Cs+, or NH+4 (Li+ and Na+ are not effective), require either Mn2+ or Fe2+ as divalent metal ion cofactors (Mg2+ is not effective), and are not protected by the substrate-metal ion complex against inhibition by the histidine-directed reagent diethyl pyrocarbonate. Chromatography of the pine xylem extracts on a quaternary amino anion-exchange resin results in the separation of four similar, but distinct, multiple product monoterpene synthases that produce sabinene, beta-phellandrene, 3-carene, and beta-pinene as the principal components, respectively. The major cyclase (phellandrene synthase) was subsequently purified by hydroxyapatite chromatography and electrophoresis. V8 proteolysis provided a peptide map significantly different from that obtained with limonene synthase from spearmint (Mentha spicata), and limited NH2-terminal sequencing of the phellandrene synthase fragments revealed no significant similarity to the deduced amino acid sequence of the angiosperm limonene synthase, the only monoterpene cyclase to be cloned and sequenced thus far. Furthermore, polyclonal antibodies raised against the angiosperm limonene synthase did not detectably cross-react with any proteins in extracts from either lodgepole pine or grand fir by immunoblotting analysis. In addition to these structural differences between cyclases from conifers and herbaceous angiosperms, the unusual pH optimum, mono- and divalent metal ion requirement, and reactivity toward histidine carbethoxylation indicate that monoterpene cyclases isolated from conifers may also have a different complement of active-site amino acid residues involved in substrate binding and catalysis than those of terpenoid cyclases previously isolated from angiosperms. << Less

  J Biol Chem 269:4012-4020(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Traumatic resin defense in Norway spruce (Picea abies): methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase.

  Faeldt J., Martin D., Miller B., Rawat S., Bohlmann J.

  Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after ... >> More

  Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense-related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase. The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae. << Less

  Plant Mol. Biol. 51:119-133(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

  Hamberger B., Hall D., Yuen M., Oddy C., Hamberger B., Keeling C.I., Ritland C., Ritland K., Bohlmann J.

  <h4>Background</h4>Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist pr ... >> More

  <h4>Background</h4>Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer.<h4>Results</h4>We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing.<h4>Conclusion</h4>We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes. << Less

  BMC Plant Biol. 9:106-106(2009) [PubMed] [EuropePMC]