The essential oil of Thymus vulgaris consists of a complex blend of mono- and sesquiterpenes that provides the plant with its characteristic aromatic odor. Within this species, several distinct chemotypes have been described, each one named after the dominating monoterpene alcohol. In this study, we identified two enzymes of the sabinene hydrate chemotype which are responsible for the biosynthesis of its major monoterpene alcohol, sabinene hydrate. Both TPS6 and TPS7 are multiproduct enzymes that formed 16 different monoterpenes and thus cover almost the whole monoterpene spectrum of the sabinene hydrate chemotype. Although the product spectra of both enzymes are similar, they form opposing enantiomers of most of their products. The monoterpene synthase TPS6 formed mostly (Z)-sabinene hydrate while the major product of TPS7 was (E)-sabinene hydrate. Incubation of the enzymes with the potential first reaction intermediates, (3R)-linalyl diphosphate and (3S)-linalyl diphosphate, revealed that the stereospecificity of TPS6 and TPS7 is determined by their ability to produce the opposing LPP enantiomers. 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, corresponding to Asn-350 in TPS6 and Ile-346 in TPS7, had a major influence on the stereochemistry of the formed products. This amino acid position is highly conserved among closely related monoterpene synthases of thyme and sage that produce a-terpineol, ?-terpinene, and cineole. Based on comparative models of mononterpene synthase protein structures with the GPP substrate docked in the active site pocket, the influence of crucial amino acid residues on the reaction mechanism is discussed.

Stereochemical mechanism of two sabinene hydrate synthases forming antipodal monoterpenes in thyme (<i>Thymus vulgaris</i>)

248th National Meeting (2014)

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