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Exploring PTDH–P450BM3 Variants for the Synthesis of Drug Metabolites
Author(s) -
Beyer Nina,
Kulig Justyna K.,
Fraaije Marco W.,
Hayes Martin A.,
Janssen Dick B.
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700470
Subject(s) - aromatization , hydroxylation , chemistry , demethylation , cofactor , enzyme , cytochrome p450 , substrate (aquarium) , oxygenase , biochemistry , cytochrome , stereochemistry , combinatorial chemistry , catalysis , biology , gene , ecology , gene expression , dna methylation
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.