Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative sesquiterpene products generated by recombinant TEAS in the single-vial GC-MS assay. The combined use of chiral and non-polar stationary phases for gas chromatography separations proved critical for resolving the numerous sesquiterpene products of TEAS for mass spectral analysis and identification. Co-injection studies with available authentic standards from both synthetic and natural sources further corroborated the assignment of several compounds, resulting in an annotated reaction mechanism accounting for their biosynthesis. Moreover, a previously undocumented farnesyl trans-cis isomerization pathway was observed.
|Number of pages||10|
|Journal||Archives of Biochemistry and Biophysics|
|State||Published - Apr 15 2006|
Bibliographical noteFunding Information:
We thank T. Kollner for the celery seed extract, J.W. Mansfield for the LTC1 clone, and R. Coates for synthetic standards of (−)-4-epi-eremophilene and (−)-prezizaene. We appreciate the insightful comments of Robert Coates, Bryan Greenhagen, and Andy Hess on various aspects of the mechanistic work. We are grateful to the National Institutes of Health for a grant that supported this work (GM54029 to Joseph P. Noel/Joseph Chappell). Paul E. O’Maille is an NIH Postdoctoral Research Fellow (GM069056). Joseph P. Noel is an investigator of the Howard Hughes Medical Institute.
- Electrophilic cyclization
- Enzyme mechanism
- Gas chromatography
- Product identification
- Terpene cyclase
- Terpene synthase
ASJC Scopus subject areas
- Molecular Biology