Purification and characterization of the monoterpene cyclase gamma-terpinene synthase from Thymus vulgaris.
The monoterpene cyclase, gamma-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 approximately 0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of Mr approximately 55,000. The protein was very hydrophobic, and possessed a pI value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 mM Mg2+; 5 mM Mn2+ could support catalysis, albeit at a much lower rate. The Km value for the substrate, geranyl pyrophosphate, was 2.6 microM. Cyclase activity was inhibited by cysteine- and histidine-directed reagents. Purified gamma-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of alpha-thujene and to lesser quantities of myrcene, alpha-terpinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol, all of which appear to be coproducts of the reaction sequence leading to gamma-terpinene. In general properties, the gamma-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.
Alonso
W R
WR
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Croteau
R
R
D019883:Alkyl and Aryl Transferases; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007525:Isoelectric Focusing; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D019684:Magnoliopsida; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022723:gamma-terpinene synthetase; D008274:Magnesium