title: Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase.
abstract: CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards beta-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 degrees C. In this system, beta-carotene was hydroxylated to beta-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low beta-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated beta-carotene at 3- and also 3'-positions, resulting in beta-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol beta-cryptoxanthin produced per nmol P450 per min.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart 70569, Germany.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Momoi
authors.forename: Ute
authors.initials: U
authors.lastname: Hofmann
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D065366:Cryptoxanthins; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology; D005288:Ferredoxins / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology; D016963:Thermus thermophilus / Q000201:enzymology; D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D065366:Cryptoxanthins; D005288:Ferredoxins; D005418:Flavodoxin; D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C079232:beta-carotene hydroxylase; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase; C022486:putidaredoxin reductase