Publications

• Crystal structure of (+)-delta-cadinene synthase from Gossypium arboreum and evolutionary divergence of metal binding motifs for catalysis.

  Gennadios H.A., Gonzalez V., Di Costanzo L., Li A., Yu F., Miller D.J., Allemann R.K., Christianson D.W.

  (+)-Delta-cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we ... >> More

  (+)-Delta-cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 A resolution and the structure of its complex with three putative Mg(2+) ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 A resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D(307)DTYD(311) motif on helix D that interacts with Mg(2+)(A) and Mg(2+)(C). However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg(2+)(B), which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg(2+)(B) ligands). Instead, DCS contains a second aspartate-rich motif, D(451)DVAE(455), that interacts with Mg(2+)(B). In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg(2+)(B) binding motif. Analyses of DCS mutants with alanine substitutions in the D(307)DTYD(311) and D(451)DVAE(455) segments reveal the contributions of these segments to catalysis. << Less

  Biochemistry 48:6175-6183(2009) [PubMed] [EuropePMC]

• Purification of (+)-delta-cadinene synthase, a sesquiterpene cyclase from bacteria-inoculated cotton foliar tissue.

  Davis E.M., Tsuji J., Davis G.D., Pierce M.L., Essenberg M.

  A sesquiterpene cyclase whose activity is induced in a glandless, bacterial blight-resistant line of cotton (Gossypium hirsutum L.) catalyses the conversion of (E,E)-farnesyl diphosphate to (+)-delta-cadinene. This enzyme was purified by a combination of salt-induced phase separation, hydroxylapat ... >> More

  A sesquiterpene cyclase whose activity is induced in a glandless, bacterial blight-resistant line of cotton (Gossypium hirsutum L.) catalyses the conversion of (E,E)-farnesyl diphosphate to (+)-delta-cadinene. This enzyme was purified by a combination of salt-induced phase separation, hydroxylapatite fractionation, hydrophobic interaction and strong anion-exchange chromatography, and denaturing polyacrylamide gel electrophoresis, followed by renaturation with Tween 80. The purified enzyme has a molecular weight of 64-65 kDa, and exhibited a single silver-staining band following electrophoresis in analytical denaturing polyacrylamide gels. Amino acid sequences of three tryptic peptides from the enzyme have been determined and are similar to known sequences in other terpene cyclases from plants. << Less

  Phytochemistry 41:1047-1055(1996) [PubMed] [EuropePMC]

• Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase.

  Steele C.L., Crock J., Bohlmann J., Croteau R.B.

  Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine, with the monoterpene ... >> More

  Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine, with the monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with farnesyl diphosphate as substrate indicated that the constitutive sesquiterpene synthases produced the same sesquiterpenes found in the oleoresin and that, in response to wounding, only two new products were synthesized, delta-cadinene and (E)-alpha-bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of sesquiterpene products. The encoded enzymes have been named delta-selinene synthase and gamma-humulene synthase based on the principal products formed; however, each enzyme synthesizes three major products and produces 34 and 52 total sesquiterpenes, respectively, thereby accounting for many of the sesquiterpenes of the oleoresin. The deduced amino acid sequence of the delta-selinene synthase cDNA open reading frame encodes a protein of 581 residues (at 67.6 kDa), whereas that of the gamma-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in identity from 43 to 46% when compared with the known sequences of monoterpene and diterpene synthases from grand fir. Although the two sesquiterpene synthases from this gymnosperm do not very closely resemble terpene synthases from angiosperm species (52-56% similarity and 26-30% identity, there are clustered regions of significant apparent homology between the enzymes of these two plant classes. The multi-step, multi-product reactions catalyzed by the sesquiterpene synthases from grand fir are among the most complex of any terpenoid cyclase thus far described. << Less

  J. Biol. Chem. 273:2078-2089(1998) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• The molecular and biochemical basis for varietal variation in sesquiterpene content in melon (Cucumis melo L.) rinds.

  Portnoy V., Benyamini Y., Bar E., Harel-Beja R., Gepstein S., Giovannoni J.J., Schaffer A.A., Burger J., Tadmor Y., Lewinsohn E., Katzir N.

  A combined chemical, biochemical and molecular study was conducted to understand the differential accumulation of volatile sesquiterpenes in melon fruits. Sesquiterpenes were present mainly in the rinds of climacteric varieties, and a great diversity in their composition was found among varieties. ... >> More

  A combined chemical, biochemical and molecular study was conducted to understand the differential accumulation of volatile sesquiterpenes in melon fruits. Sesquiterpenes were present mainly in the rinds of climacteric varieties, and a great diversity in their composition was found among varieties. Sesquiterpenes were generally absent in non-climacteric varieties. Two climacteric melon varieties, the green-fleshed 'Noy Yizre'el', and the orange-fleshed 'Dulce' were further examined. In 'Noy Yizre'el' the main sesquiterpenes accumulated are delta-cadinene, gamma-cadinene and alpha-copaene, while alpha-farnesene is the main sesquiterpene in 'Dulce'. Sesquiterpene synthase activities, mainly restricted to rinds of mature fruits, were shown to generate different sesquiterpenes in each variety according to the compositions found in rinds. EST melon database mining yielded two novel cDNAs coding for members of the Tps gene family termed CmTpsNY and CmTpsDul respectively, that are 43.2% similar. Heterologous expression in E. coli of CmTpsNY produced mainly delta-copaene, alpha-copaene, beta-caryophyllene, germacrene D, alpha-muurolene, gamma-cadinene, delta-cadinene, and alpha-cadinene, while CmTpsDul produced alpha-farnesene only. CmTpsNY was mostly expressed in 'Noy Yizre'el' rind while CmTpsDul expression was specific to 'Dulce' rind. None of these genes was expressed in rinds of the non-climacteric 'Tam Dew' cultivar. Our results indicate that different sesquiterpene synthases encoded by different members of the Tps gene family are active in melon varieties and this specificity modulates the accumulation of sesquiterpenes. The genes are differentially transcriptionally regulated during fruit development and according to variety and are likely to be associated with chemical differences responsible for the unique aromas of melon varieties. << Less

  Plant Mol. Biol. 66:647-661(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis.

  Chen X.-Y., Chen Y., Heinstein P., Davisson V.J.

  In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection. A Gossypium arboreum cell suspension culture which produces the sesquiterpene phytoalexin gossypol showed a time-dependent 10-fold increase in a 1.9-kb mRNA in response to a challenge by a preparation ... >> More

  In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection. A Gossypium arboreum cell suspension culture which produces the sesquiterpene phytoalexin gossypol showed a time-dependent 10-fold increase in a 1.9-kb mRNA in response to a challenge by a preparation from Verticillium dahliae. The mRNA prepared from these elicited cultures was used to isolated two cDNA clones that contain open frames coding for proteins of 554 amino acids with M(r) 64,096 and 64,118. The encoded protein shows a significant degree of sequence identity with the other known plant terpene cyclases. Western blot analyses with a cross-reactive monoclonal antibody from a related sesquiterpene synthase in Nicotiana tabacum showed a time-dependent increase of a 65-kDa protein which reached a maximal level 24 h post elicitor treatment. The encoded protein from the pXC1 cDNA was produced in Escherichia coli and purified by affinity column chromatography. The enzymatic properties of this protein were identified by a radiochemical assay for cyclization of farnesyldiphosphate and a product structure was assigned by GC-MS, chiral phase GC, and NMR analyses as (+)-delta-cadinene. The fungal-elicited production of a (+)-delta-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton. << Less

  Arch. Biochem. Biophys. 324:255-266(1995) [PubMed] [EuropePMC]