Purification and characterization of the monoterpene cyclase γ-terpinene synthase from Thymus vulgaris

Abstract

The monoterpene cyclase, γ-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h · mg protein (turnover number of ~0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of M r ~ 55,000. The protein was very hydrophobic, and possessed a p I value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 m m Mg 2+; 5 m m Mn 2+ could support catalysis, albeit at a much lower rate. The K m value for the substrate, geranyl pyrophosphate, was 2.6 μ m. Cyclase activity was inhibited by cysteineand histidine-directed reagents. Purified γ-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of α-thujene and to lesser quantities of myrcene, α-terpinene, limonene, linalool, terpinen-4-ol, and α-terpineol, all of which appear to be coproducts of the reaction sequence leading to γ-terpinene. In general properties, the γ-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.