Identification of Mutant Asp251Gly/Gln307His of Cytochrome P450 BM3 for the Generation of Metabolites of Diclofenac, Ibuprofen and Tolbutamide

• Published in: Chemistry : a European journal Vol. 18; no. 12; pp. 3582 - 3588
• Main Authors: Tsotsou, Georgia E., Sideri, Anastasia, Goyal, Abhineet, Di Nardo, Giovanna, Gilardi, Gianfranco
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 19-03-2012; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: Asparagine - chemistry; Asparagine - genetics; Bacillus megaterium - metabolism; Bacteria; Bacteriology; Catalysis; Chemistry; Cytochrome; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochromes P450; Diclofenac; Diclofenac - analogs & derivatives; Diclofenac - chemistry; Diclofenac - metabolism; directed evolution; Drug Discovery; Drugs; Escherichia coli - genetics; Escherichia coli - metabolism; Glutamine - chemistry; Glutamine - genetics; Glycine - chemistry; Glycine - genetics; Histidine - chemistry; Histidine - genetics; Humans; Hydroxylation; Ibuprofen; Ibuprofen - analogs & derivatives; Ibuprofen - chemistry; Ibuprofen - metabolism; Metabolites; NADP - metabolism; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - genetics; Oxidation-Reduction; Substrates; tolbutamide; Tolbutamide - chemistry; Tolbutamide - metabolism
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201102470

The soluble, catalytically self‐sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error‐prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and KM values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4′‐hydroxydiclofenac, 2‐hydroxyibuprofen and 4‐hydroxytolbutamide, respectively.
Soluble bacterial P450: The mutant Asp251Gly/Gln307His of the bacterial cytochrome P450 BM3 shows new catalytic activity towards diclofenac, ibuprofen and tolbutamide. It can be used to synthesise the metabolites of these drugs normally produced by the 2C subfamily of human cytochromes P450 (see scheme).