title: Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans.
abstract: Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.
authors.affiliation: Department of Pharmaceutical Biology, University Centre for Pharmacy, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.
authors.forename: G A
authors.initials: GA
authors.lastname: Molog
authors.forename: U
authors.initials: U
authors.lastname: Empt
authors.forename: S
authors.initials: S
authors.lastname: Kuhlmann
authors.forename: W
authors.initials: W
authors.lastname: van Uden
authors.forename: N
authors.initials: N
authors.lastname: Pras
authors.forename: A W
authors.initials: AW
authors.lastname: Alfermann
authors.forename: M
authors.initials: M
authors.lastname: Petersen
mesh_terms: D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003574:Cytochrome c Group / Q000494:pharmacology; D004365:Drugs, Chinese Herbal; D019597:Flax / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D017705:Lignans / Q000096:biosynthesis*; D008861:Microsomes / Q000201:enzymology; D009243:NAD / Q000008:administration & dosage; D009249:NADP / Q000008:administration & dosage; D011034:Podophyllotoxin / Q000031:analogs & derivatives* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D003574:Cytochrome c Group; D004365:Drugs, Chinese Herbal; D017705:Lignans; D009243:NAD; D009249:NADP; C014451:deoxypodophyllotoxin; D003577:Cytochrome P-450 Enzyme System; D011034:Podophyllotoxin