Stereochemical mechanism of two sabinene hydrate synthases forming antipodal monoterpenes in thyme (Thymus vulgaris).

The essential oil of Thymus vulgaris consists of a complex blend of mono- and sesquiterpenes that provides the plant with its characteristic aromatic odor. Several chemotypes have been described for thyme. In this study, we identified two enzymes of the sabinene hydrate chemotype which are responsible for the biosynthesis of its major monoterpene alcohols, (1S,2R,4S)-(Z)-sabinene hydrate and (1S,2S,4R)-(E)-sabinene hydrate. Both TPS6 and TPS7 are multiproduct enzymes that formed 16 monoterpenes and thus cover almost the whole monoterpene spectrum of the chemotype. Although the product spectra of both enzymes are similar, they form opposing enantiomers of their chiral products. Incubation of the enzymes with the potential reaction intermediates revealed that the stereospecificity of TPS6 and TPS7 is determined by the formation of the first intermediate, linalyl diphosphate. Since TPS6 and TPS7 shared an amino acid sequence identity of 85%, a mutagenesis study was employed to identify the amino acids that determine the stereoselectivity. One amino acid position had a major influence on the stereochemistry of the formed products. Based on comparative models of TPS6 and TPS7 protein structures with the GPP substrate docked in the active site pocket, the influence of this amino acid residue on the reaction mechanism is discussed.

Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis.

The aroma, flavor and pharmaceutical value of cultivated oregano (Origanum vulgare L.) is a consequence of its essential oil which consists mostly of monoterpenes and sesquiterpenes. To investigate the biosynthetic pathway to oregano terpenes and its regulation, we identified and characterized seven terpene synthases, key enzymes of terpene biosynthesis, from two cultivars of O. vulgare. Heterologous expression of these enzymes showed that each forms multiple mono- or sesquiterpene products and together they are responsible for the direct production of almost all terpenes found in O. vulgare essential oil. The correlation of essential oil composition with relative and absolute terpene synthase transcript concentrations in different lines of O. vulgare demonstrated that monoterpene synthase activity is predominantly regulated on the level of transcription and that the phenolic monoterpene alcohol thymol is derived from gamma-terpinene, a product of a single monoterpene synthase. The combination of heterologously-expressed terpene synthases for in vitro assays resulted in blends of mono- and sesquiterpene products that strongly resemble those found in vivo, indicating that terpene synthase expression levels directly control the composition of the essential oil. These results will facilitate metabolic engineering and directed breeding of O. vulgare cultivars with higher quantity of essential oil and improved oil composition.