Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Damsten, Micaela C.; Vugt-Lussenburg, Barbara M.A. van; Zeldenthuis, Tineke; Vlieger, Jon S. B. de; Commandeur, Jan N. M.; Vermeulen, Nico

doi:10.1016/j.cbi.2007.09.007

Cited by 86 publications

(131 citation statements)

References 24 publications

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“…Drugs tested were acetaminophen, diclofenac, and clozapine (CLZ), and formation of reactive intermediates was analyzed by measurement of GSH conjugates. For all drugs tested, most stable metabolites and reactive intermediates were produced at much higher activity by the CYP102A1 mutants than by human and rat liver microsomes, supporting their potential for use in characterization of toxicologically relevant metabolites (Damsten et al, 2008).…”

Section: Introductionmentioning

confidence: 68%

“…Oxidative pathways of metabolism of CLZ by cytochrome P450 and nonenzymatic and enzymatic conjugation reactions of reactive CLZ nitrenium ion by GSH and GST: a, N-demethylation; b, N-oxidation; c, oxidative opening of piperazine ring; d, dehydrogenation to nitrenium ion. Although CYP102A1 M11H was previously shown to produce high levels of CLZ metabolites, the most abundant metabolites appeared to be N-demethylclozapine and CLZ N-oxide (Damsten et al, 2008;Dragovic et al, 2010). As a consequence, GSH conjugates derived from these metabolites were also produced, which strongly complicates isolation of CG-4 and CG-5.…”

Section: Introductionmentioning

confidence: 98%

“…By combinations of site-directed and random mutagenesis, many CYP102A1 mutants have been obtained that are able to convert drugs and drug-like molecules to human-relevant metabolites (van Vugt-Lussenburg et al, 2007;Yun et al, 2007;Sawayama et al, 2009;Kim et al, 2011). In our previous work, four mutants of CYP102A1 were evaluated as biocatalysts for the bioactivation of several drugs to reactive intermediates (Damsten et al, 2008). Drugs tested were acetaminophen, diclofenac, and clozapine (CLZ), and formation of reactive intermediates was analyzed by measurement of GSH conjugates.…”

Section: Introductionmentioning

confidence: 99%

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Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Rea¹,

Dragović²,

Boerma³

et al. 2011

Drug Metab Dispos

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ABSTRACT:In the present study, a site-saturation mutagenesis library of drugmetabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by 1 H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.

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Section: Introductionmentioning

confidence: 68%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Rea¹,

Dragović²,

Boerma³

et al. 2011

Drug Metab Dispos

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“…The direct use of human cytochrome P450 is limited by the need of a redox partner and the poor stability and activity. However, bacterial counterparts such as cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, can be directly used [3] or engineered to metabolise drugs and to produce the same metabolites as the human enzymes [4][5][6][7][8][9][10][11][12][13][14]. This protein is a selfsufficient fatty acids monoxygenase containing a NADPH-dependent reductase (BMR) and a P450 catalytic domain (BMP) fused in a single polypeptidic chain [15,16].…”

Section: Introductionmentioning

confidence: 99%

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Nardo

Dell'Angelo

Catucci

et al. 2016

Archives of Biochemistry and Biophysics

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This paper reports the structure of the double mutant Asp251Gly/Gln307His (here called A2), generated by random mutagenesis, is able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. Here, we report the crystal structure of the heme domain of the mutant in the substrate-free form and in complex with the substrate N-palmitoylglycine, together with its impact on thermal stability, reduction potential and electron transfer.The substrate-free structure adopts a conformation more similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop, part of the substrate access channel. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on Ihelix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules.Differential scanning calorimetry shows a population destabilized by 2.2°C compared to the wild type protein, probably reflecting the increased flexibility of part of the protein compared to WT. Moreover, a shift of the heme reduction potential by 50 mV toward positive values, a 2-folds higher first electron transfer rate in the absence of oxygen and a 4-folds higher NADPH consumption rate in the presence of oxygen as the only electron acceptors, when compared to wild type protein.The data demonstrate that a single mutation far away from the active site is able to trigger an increase in protein flexibility in a key region of the substrate access channel, shifting the conformational equilibrium toward the closed form of the protein that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.

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“…Rates (pmol demethylated clozapine formed per min) were estimated using a standard curve of the substrate (clozapine), assuming that the extinction coefficients of the substrate and metabolite (demethylated clozapine) are comparable. 31 To verify Michaelis-Menten characteristics (that is, time, substrate and enzyme dependence) of the metabolism of clozapine by CYP1A2, a full kinetic analysis with WT was performed. For this purpose, 100 nM of CYP1A2 was used per incubation and a gradient of 7 clozapine concentrations (p500 mM).…”

Section: -Cyano-7-ethoxycoumarin O-dealkylationmentioning

confidence: 99%

Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

et al. 2010

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Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Cited by 86 publications

References 24 publications

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

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