title: Purification and characterization of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti.
abstract: The sesquiterpene cyclase, aristolochene synthase, has been purified from Penicillium roqueforti by gel filtration and anion-exchange chromatography. Isolation was facilitated by a change in the elution behavior of the enzyme during gel filtration at different steps in the purification. The purified enzyme had a specific activity of 70 nmol/min/mg protein. The molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was Mr 37,000. The native molecular weight as determined by gel filtration chromatography was Mr 48,000. The requirement for Mg2+ could be partially substituted with 0.01 mM Mn2+, but higher concentrations were inhibitory. Pyrophosphate, a competitive inhibitor of most terpene cyclases, had no effect on enzyme activity up to a concentration of 5.0 mM. The maximum activity was observed between pH 6.25 and pH 7.50, and the Km for farnesyl pyrophosphate was 0.55 +/- 0.06 microM.
authors.affiliation: Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Illinois 61604.
authors.forename: T M
authors.initials: TM
authors.lastname: Hohn
authors.forename: R D
authors.initials: RD
authors.lastname: Plattner
mesh_terms: D002412:Cations; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D011756:Diphosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D012717:Sesquiterpenes
chemical_list: D002412:Cations; D011756:Diphosphates; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D008345:Manganese; C004808:farnesyl pyrophosphate; D007535:Isomerases; C060050:aristolochene synthase; D008274:Magnesium