Tris(tetrabutylammonium) hydrogen pyrophosphate. A new reagent for the preparation of allylic pyrophosphate esters

Dixit, Vandana; Laskovics, F. M.; Noall, Wendy I.; Poulter, C. Dale

doi:10.1021/jo00322a060

Cited by 53 publications

(22 citation statements)

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“…Spectra of the putative germacrene C was consistent with the germacrene C structure. The following chemical shifts (␦) were recorded for 13 C NMR (see Fig. 5 Germacrene B was identified by GC-MS comparison with the authentic standard.…”

Section: Resultsmentioning

confidence: 99%

Germacrene C synthase fromLycopersicon esculentumcv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

Colby

Crock

Dowdle-Rizzo

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

121

100

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Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, ␤-caryophyllene, ␣-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3 H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (M r ‫؍‬ 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton ␦-cadinene synthase (50% identity).

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Section: Resultsmentioning

confidence: 99%

Germacrene C synthase fromLycopersicon esculentumcv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

Colby

Crock

Dowdle-Rizzo

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

121

100

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“…Synthetic procedures [1][2][3] H]GPP (56 Ci/mol) was prepared by literature procedures [16], as was 3-azaGPP [17]. 2-FGPP (7) [18] and (±)-2-FLPP (8) [19] were prepared as outlined in Schemes 1 and 2.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

Karp

Zhao

Santhamma

et al. 2007

Archives of Biochemistry and Biophysics

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The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)-and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (−)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)-and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.

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“…[4-14 C]IPP (54 Ci/mol) was purchased from DuPont/NEN. DMAPP was synthesized as previously described (19), as were GPP (20) and FPP (21). Terpenol standards were from our own collection.…”

Section: Methodsmentioning

confidence: 99%

Geranyl diphosphate synthase: Cloning, expression, and characterization of this prenyltransferase as a heterodimer

Burke

Wildung

Croteau

1999

Proc. Natl. Acad. Sci. U.S.A.

199

145

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Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer. monoterpene biosynthesis ͉ isoprenoid biosynthesis

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Tris(tetrabutylammonium) hydrogen pyrophosphate. A new reagent for the preparation of allylic pyrophosphate esters

Cited by 53 publications

References 0 publications

Germacrene C synthase fromLycopersicon esculentumcv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

Germacrene C synthase fromLycopersicon esculentumcv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

Geranyl diphosphate synthase: Cloning, expression, and characterization of this prenyltransferase as a heterodimer

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