(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.
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 (3)H- and (2)H-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.
Prosser
Ian
I
Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Bristol, BS41 9AF, Long Ashton, UK.
Phillips
Andy L
AL
Gittings
Simon
S
Lewis
Mervyn J
MJ
Hooper
Antony M
AM
Pickett
John A
JA
Beale
Michael H
MH
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D036624:Solidago / Q000201:enzymology*
D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases; C462022:germacrene A synthase, Cichorium intybus