γ-Terpinene synthase of Thymus vulgaris

Abstract

Thyme is a plant genus comprising over 300 species within the Lamiaceae family. For medicine, especially for the treatment of respiratory diseases, the essential oil is the most important ingredient. It shows antibacterial and antiviral effects. Essential oils are complex mixtures composed of active and inactive compounds [1, 2]. In Thymus sepyllum (Ts) and six Thymus species the essential oils were isolated by hydrodestillation and analyzed by GC-MS. The γ-terpinene was identified as a characteristic monoterpene in all essential oils, albeit in varying concentrations. It is a naturally occurring monoterpene and a major component in most essential oils of citrus fruits and many aromatic plants. γ-Terpinene is formed through cyclisation of geranyl diphosphate (GPP) by γ-terpinene synthase which belong to the monoterpene cyclase family [3]. Traces of several other monoterpenoids were formed in addition to γ-terpinene (product promiscuity). Using primers derived from Origanum vulgare, we here isolated and sequenced γ-terpinene synthase cDNAs (TPS) from Thymus vulgaris (Tv), Thymus serpyllum (Ts), Thymus x citriodorus (Txc) and Thymus caespititius (Tc). Sequence data were used to study the chemical-taxonomic relationship between the four species. The bastard plant Txc showed a close relationship to Tv, whereas Tc γ-terpinene synthase was closely related to that of Origanum vulgare [4]. The Tv γ-terpinene synthase gene 1 (TvTPS1), which encoded for a protein of 596 amino acids was expressed as a recombinant protein in E. coli. We succeeded in overproduction, purification and crystallization of the TPS from Thymus vulgaris for the first time.