title: P450BM3fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.
abstract: To facilitate the wider application of the NADPH-dependent P450BM3,we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3driven by NADPH. The results suggest that PTDH-P450BM3is an attractive system for use in biocatalytic and drug metabolism studies.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors.forename: Nina
authors.initials: N
authors.lastname: Beyer
authors.affiliation: Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 43183, Sweden.
authors.forename: Justyna K
authors.initials: JK
authors.lastname: Kulig
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors.forename: Anette
authors.initials: A
authors.lastname: Bartsch
authors.affiliation: Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 43183, Sweden.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Hayes
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. m.w.fraaije@rug.nl.
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D001410:Bacillus megaterium / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009853:Omeprazole / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D017905:Phosphites / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077154:Rosiglitazone; D013379:Substrate Specificity; D045162:Thiazolidinediones / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007850:Lauric Acids; D017905:Phosphites; D011993:Recombinant Fusion Proteins; D045162:Thiazolidinediones; D000077154:Rosiglitazone; C030358:lauric acid; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; C429539:NAD phosphite oxidoreductase; D009853:Omeprazole
keywords: Drug metabolites; Enzyme catalysis; Fatty acids; NADPH regeneration; Oxidation; Protein engineering