2012
DOI: 10.1002/chem.201102470
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Identification of Mutant Asp251Gly/Gln307His of Cytochrome P450 BM3 for the Generation of Metabolites of Diclofenac, Ibuprofen and Tolbutamide

Abstract: 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 du… Show more

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Cited by 32 publications
(34 citation statements)
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“…This last feature is usually associated to the uncoupling of reducing equivalents for reactive oxygen species production rather than for product formation found in cytochromes P450 [1,44]. This may be the reason why the turnover rate for drugs in A2 mutant is much lower than the NADPH consumption rate, suggesting a high level of uncoupling [9]. Furthermore, these data confirm that a slower electron transfer is usually associated to a higher level of coupling [45][46][47].…”
Section: Impact Of Structure On P450 Bmp A2 Functional Propertiessupporting
confidence: 52%
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“…This last feature is usually associated to the uncoupling of reducing equivalents for reactive oxygen species production rather than for product formation found in cytochromes P450 [1,44]. This may be the reason why the turnover rate for drugs in A2 mutant is much lower than the NADPH consumption rate, suggesting a high level of uncoupling [9]. Furthermore, these data confirm that a slower electron transfer is usually associated to a higher level of coupling [45][46][47].…”
Section: Impact Of Structure On P450 Bmp A2 Functional Propertiessupporting
confidence: 52%
“…Since the mutant A2 has been shown to have new catalytic abilities toward diclofenac, ibuprofen and tolbutamide [9], co-crystallization experiments were also performed by complexing the enzyme with the three drugs at saturating concentrations before crystallization and monitoring the spin shift associated to the drug binding. For the A2-diclofenac complex, diffraction data were collected at 2.0 Å resolution and the structure solved.…”
Section: Crystal Structures Of the Heme Domain Of A2mentioning
confidence: 99%
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“…The recombinant expression of CYP2C9 in fission yeast strain CAD68 resulted in an efficient formation of product 5a (468 mg/l) after the optimization of the pH value, the glucose concentration, and the establishment of a favorable host organism for the hydroxylation of substrate 5 (Drǎgan et al, 2011). The engineering of BM3 toward the metabolism of drugs resulted in the BM3 mutant Asp251Gly/ Gln307His, capable of the metabolism of drug 5 to product 5a in vitro (Tsotsou et al, 2012). This BM3 mutant was also shown to produce 2-hydroxyibuprofen (7a) from ibuprofen (7) (Tsotsou et al, 2012); however, experiments were only done in vitro or in a microtiter plate.…”
Section: Discussionmentioning
confidence: 99%