Isolation, characterization, and mechanistic studies of (-)-alpha-gurjunene synthase from Solidago canadensis.
The leaves of the composite Solidago canadensis (goldenrod) were shown to contain (-)-alpha-gurjunene synthase activity. This sesquiterpene is likely to be the precursor for cyclocolorenone, a sesquiterpene ketone present in high amounts in S. canadensis leaves. (-)-alpha-Gurjunene synthase was purified to apparent homogeneity (741-fold) by anion-exchange chromatography (on several matrices), dye ligand chromatography, hydroxylapatite chromatography, and gel filtration. Chromatography on a gel filtration matrix indicated a native molecular mass of 48 kDa, and SDS-PAGE showed the enzyme to be composed of one subunit with a denatured mass of 60 kDa. Its maximum activity was observed at pH 7.8 in the presence of 10 mM Mg2+ and the KM value for the substrate farnesyl diphosphate was 5.5 microM. Over a range of purification steps (-)-alpha-gurjunene and (+)-gamma-gurjunene synthase activities copurified. In addition, the product ratio of the enzyme activity under several different assay conditions was always 91% (-)-alpha-gurjunene and 9% (+)-gamma-gurjunene. This suggests that the formation of these two structurally related products is catalyzed by one enzyme. For further confirmation, we carried out a number of mechanistic studies with (-)-alpha-gurjunene synthase, in which an enzyme preparation was incubated with deuterated substrate analogues. Based on mass spectrometry analysis of the products formed, a cyclization mechanism was postulated which makes it plausible that the synthase catalyzes the formation of both sesquiterpenes.
Schmidt
C O
CO
Institut für Organische Chemie, Universität Hamburg, Hamburg, D-20146, Germany.
Bouwmeester
H J
HJ
Bülow
N
N
König
W A
WA
D019659:Asteraceae / Q000201:enzymology*; D003521:Cyclopropanes / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism
D003521:Cyclopropanes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C007186:cyclocolorenone; C004808:farnesyl pyrophosphate