Journal
PHYTOCHEMISTRY
Volume 60, Issue 7, Pages 691-702Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/S0031-9422(02)00165-6
Keywords
farnesyl diphosphate; germacrene A; goldenrod; Solidago canadensis; sesquiterpene synthase
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Funding
- Biotechnology and Biological Sciences Research Council [BBS/E/C/00004162] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BBS/E/C/00004162] Funding Source: Medline
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Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of H-3- and H-2-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco. (C) 2002 Elsevier Science Ltd. All rights reserved.
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