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

• 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
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja020762p

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.