1. Exploring PTDH-P450BM3 variants for the synthesis of drug metabolites
- Author
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Justyna Kulig, Nina Beyer, Dick B. Janssen, Marco W. Fraaije, Martin A. Hayes, and Biotechnology
- Subjects
0301 basic medicine ,Oxygenase ,Cytochrome ,030226 pharmacology & pharmacy ,Biochemistry ,Cofactor ,Mass Spectrometry ,Enzyme catalysis ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,enzyme fusion ,NADH, NADPH Oxidoreductases ,Molecular Biology ,Chromatography, High Pressure Liquid ,Demethylation ,NADPH-Ferrihemoprotein Reductase ,chemistry.chemical_classification ,biology ,Molecular Structure ,Chemistry ,enzymatic conversion ,Organic Chemistry ,Aromatization ,Genetic Variation ,PTDH-P450 variants ,drug metabolite synthesis ,030104 developmental biology ,Enzyme ,Pharmaceutical Preparations ,Mutation ,biology.protein ,Biocatalysis ,Molecular Medicine ,Drug metabolism - Abstract
The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.
- Published
- 2018
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