Biosynthetic potential of sesquiterpene synthases: Alternative products of tobacco 5-epi-aristolochene synthase

Abstract

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.

Interception of the Enzymatic Conversion of Farnesyl Diphosphate to 5‐Epi‐Aristolochene by Using a Fluoro Substrate Analogue: 1‐Fluorogermacrene A from (2E,6Z)‐6‐Fluorofarnesyl Diphosphate

“…[37] In some instances, these eudesmane hydrocarbons have been reported as co-products of reactions catalyzed by wild-type TEAS [38] and by mutants of aristolochene synthase. [4,39] Hz) and 19b (δ F =-183.90, J=48.9, 22.9 Hz) arise from axial/axial and axial/equatorial couplings to the protons on C2, in support of the 1α (axial) configurations of the fluoro substituents shown in Scheme 7.…”

Evolution of a Complex Locus for Terpene Biosynthesis in Solanum

“…However, in vitro testing has shown that several tomato TPSs from other clades that use trans-prenyl diphosphates in vivo are capable of using cis-prenyl diphosphate substrates in vitro (Falara et al, 2011), and some sesquiterpene synthases from tobacco can use cis-trans FPP in addition to all-trans FPP as substrates (O'Maille et al, 2006). More recently, a CPT gene that catalyzes the head-to-middle condensation of two DMAPP molecules to produce lavendulyl diphosphate has been described in lavender (Lavendula x. intermedia) (Demissie et al, 2013).…”

Unearthing the roots of the terpenome

“…In other examples, taxadiene synthase catalyzes the cyclization of 6-fluorogeranylgeranyl diphosphate to generate a mixture of 7-fluoroverticillenes [39], and aristolochene synthase catalyzes the cyclization of 2-fluorofarnesyl diphosphate to form 2-fluorogermacrene A [40]. 5-Epi-aristolochene synthase, for which the stereochemical details of the cyclization mechanism have been recently outlined [41], generates 25 sesquiterpene products [42], and mutagenesis studies targeting evolutionarily variable residues in and surrounding the active site show that product arrays can be altered by design [43].…”