title: CYP119 plus a Sulfolobus tokodaii strain 7 ferredoxin and 2-oxoacid:ferredoxin oxidoreductase constitute a high-temperature cytochrome P450 catalytic system.
abstract: The cytochrome P450 superfamily of enzymes catalyzes a broad range of oxidative processes involved in the metabolism of fatty acids, biosynthesis of sterols, and elimination of drugs and xenobiotics. Application of the unique properties of P450 enzymes as fine biocatalysts in biotechnology is limited due to their thermal instability and the requirement for auxiliary electron-donor proteins and cofactors. CYP119, a thermophilic P450 enzyme from Sulfolobus solfataricus, was characterized some time ago, but no high-temperature redox partners have been available for it. Here we report reconstitution of CYP119 with a novel high-temperature electron-donor system consisting of a ferredoxin and 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii strain 7 that, unlike all other known P450 electron-donor partners, utilizes coenzyme-A and pyruvic acid rather than NADH or NADPH as the source of electrons. The oxidation of lauric acid by the reconstituted system increased 16-fold as the temperature increased from 25 to 70 degrees C and was functional for more than 30 min at the higher temperature. This first in vitro high-temperature P450 catalytic system is a key step in the development of practical high-temperature monooxygenase systems.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Puchkaev
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Wakagi
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D019843:Archaeal Proteins; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D005288:Ferredoxins; D007850:Lauric Acids; D009243:NAD; C030358:lauric acid; D009249:NADP; D019289:Pyruvic Acid; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase