Purification and characterization of the sesquiterpene cyclase patchoulol synthase from Pogostemon cablin.
The sesquiterpene cyclase, patchoulol synthase, from Pogostemon cablin (patchouli) leaves was purified to apparent homogeneity by chromatofocusing, anion exchange, gel permeation, and hydroxylapatite chromatography. The enzyme showed a maximum specific activity of about 20 nmol/min/mg protein, and a native molecular weight of 80,000 as determined by gel permeation chromatography. The protein was very hydrophobic, as judged by chromatographic behavior on several matrices, and possessed a pI value of about 5.0, as determined by isoelectric and chromatofocusing. SDS-PAGE showed the enzyme to be composed of two apparently identical subunits of Mr approximately 40,000. Maximum activity was observed at pH 6.7 in the presence of Mg2+ (Km approximately 1.7 mM); other divalent metal ions were ineffective in promoting catalysis. The Km value for the substrate, farnesyl pyrophosphate, was 6.8 microM. Patchoulol synthase copurified with the ability to transform farnesyl pyrophosphate to cyclic olefins (alpha- and beta-patchoulene, alpha-bulnesene, and alpha-guiaene) and this observation, plus evidence based on differential inhibition and inactivation studies, suggested that these structurally related products are synthesized by the same cyclase enzyme. In general properties, the patchoulol synthase from patchouli leaves resembles fungal sesquiterpene olefin cyclases except for the ability to synthesize multiple products, a property more typical of monoterpene cyclases of higher plant origin.
Munck
S L
SL
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Croteau
R
R
D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007202:Indicators and Reagents; D007535:Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*
D006882:Hydroxyapatites; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D017886:Durapatite; D007535:Isomerases; C065995:patchoulol synthase