Germacrene A Is a Product of the Aristolochene Synthase-Mediated Conversion of Farnesylpyrophosphate to Aristolochene

The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that s...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2002-10, Vol.124 (39), p.11636-11641
Main Authors: Calvert, Melanie J, Ashton, Peter R, Allemann, Rudolf K
Format: Article
Language:English
Subjects:
Analytical biochemistry: general aspects, technics, instrumentation
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Circular Dichroism
Escherichia coli - enzymology
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gas Chromatography-Mass Spectrometry
Isomerases - biosynthesis
Isomerases - genetics
Isomerases - metabolism
Kinetics
Molecular biophysics
Penicillium - enzymology
Penicillium - metabolism
Polyisoprenyl Phosphates - metabolism
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sesquiterpenes - metabolism
Spectroscopy : techniques and spectras
Tyrosine - metabolism
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Summary:The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that significant amounts of germacrene A (7.5% of the total amount of products) are indeed released from wild-type aristolochene synthase (AS) from Penicillium roqueforti. Germacrene A was identified through direct GC-MS comparison to an authentic sample and through production of β-elemene in a thermal Cope rearrangement. AS also produced a small amount of valencene through deprotonation of C6 rather than C8 in the final step of the reaction. On the basis of the X-ray structure of AS, Tyr 92 was postulated to be the active-site acid responsible for protonation of germacrene A (Caruthers, J. M.; Kang, I.; Rynkiewicz, M. J.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2000, 275, 25533−25539). The CD spectra of a mutant protein, ASY92F, in which Tyr 92 was replaced by Phe, and of AS were very similar. ASY92F was approximately 0.1% as active as nonmutated recombinant AS. The steady-state kinetic parameters were measured as 0.138 min-1 and 0.189 mM for k cat and K M, respectively. Similar to a mutant protein of 5-epi-aristolochene (Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc. 2000, 122, 1861−1866), the mutant released significant amounts of germacrene A (∼29%). ASY92F also produced various amounts of a further five hydrocarbons of molecular weight 204, valencene, β-(E)-farnesene, α- and β-selinene, and selina-4,11-diene.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja020762p