Roles of key active-site residues in flavocytochrome P450 BM3

Noble, Michael A.; Miles, Caroline S.; Chapman, Stephen K; Lysek, D.A.; Mackay, Angela C.; Reid, Graeme A.; Hanzlik, Robert P.; Munro, Andrew W.

doi:10.1042/0264-6021:3390371

Cited by 159 publications

(212 citation statements)

References 19 publications

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“…These values agree with previous measurements. 52, 66 Subsequently, we measured the rate of 14,15-EET formation from AA and 17,18-EEQ formation from EPA. The rate of formation of 14,15-EET was determined to be 1.10 ± 0.21 μmol/min/nmol BM3 , and for 17,18-EEQ to be 2.57 ± 0.09 μmol/min/nmol BM3 , which correlates well to previous data.…”

Section: Resultsmentioning

confidence: 99%

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Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase

et al. 2016

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Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an ω-6 polyunsaturated fatty acid (PUFA), into anti-inflammatory epoxide metabolites. It also epoxidizes other PUFAs such as docosahexaenoic acid (DHA), linoleic acid (LA), and eicosapentaenoic acid (EPA). Herein, we have performed detailed thermodynamic and kinetic analyses to determine how DHA, LA and EPA modulate AA metabolism by CYP2J2. We use the Nanodisc (ND) system to stabilize CYP2J2 and its redox partner CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, while LA only moderately inhibits and EPA exhibits insignificant inhibition. We also characterized the binding of these molecules using ebastine competitive binding assays and show that DHA binds significantly tighter to CYP2J2 as compared to AA, EPA, or LA. Furthermore, we utilize a combined approach of molecular dynamics (MD) simulations and docking to predict key residues mediating the tight binding of DHA. We show that although all the tested fatty acids form similar contacts to the active site residues, the affinity of DHA binding to CYP2J2 is tighter due to the interaction of DHA with residues Arg-321, Thr-318 and Ser-493. To demonstrate the importance of these residues in binding, we mutated these residues to make two mutant variants—CYP2J2-T318A and CYP2J2-T318V/S493A. Both of these variants showed weaker binding affinity to DHA and AA compared to the WT and the stronger inhibition of AA by DHA in the WT is mitigated in these mutants. Therefore, using a combined experimental and MD simulations approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA and LA is asymmetric due to tighter binding of DHA to select residues in the active site.

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Section: Resultsmentioning

confidence: 99%

“…The rate of formation of 14,15-EET was determined to be 1.10 ± 0.21 μmol/min/nmol BM3 , and for 17,18-EEQ to be 2.57 ± 0.09 μmol/min/nmol BM3 , which correlates well to previous data. 52, 66 …”

Section: Resultsmentioning

confidence: 99%

Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase

et al. 2016

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“… ND no detectable peak at 450 nm, NQ not quantifiable owing to the absence of a peak at 450 nm a CYP102A1 M11 containing R47L, E64G, F81I, F87V, E143G, L188Q, E267V, and G415S [9]…”

Section: Resultsmentioning

confidence: 99%

Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11

et al. 2011

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CYP102A1, originating from Bacillus megaterium, is a highly active enzyme which has attracted much attention because of its potential applicability as a biocatalyst for oxidative reactions. Previously we developed drug-metabolizing mutant CYP102A1 M11 by a combination of site-directed and random mutagenesis. CYP102A1 M11 contains eight mutations, when compared with wild-type CYP102A1, and is able to produce human-relevant metabolites of several pharmaceuticals. In this study, active-site residue 87 of drug-metabolizing mutant CYP102A1 M11 was mutated to all possible natural amino acids to investigate its role in substrate selectivity and regioselectivity. With alkoxyresorufins as substrates, large differences in substrate selectivities and coupling efficiencies were found, dependent on the nature of residue 87. For all combinations of alkoxyresorufins and mutants, extremely fast rates of NADPH oxidation were observed (up to 6,000 min−1). However, the coupling efficiencies were extremely low: even for the substrates showing the highest rates of O-dealkylation, coupling efficiencies were lower than 1%. With testosterone as the substrate, all mutants were able to produce three hydroxytestosterone metabolites, although with different activities and with remarkably different product ratios. The results show that the nature of the amino acid at position 87 has a strong effect on activity and regioselectivity in the drug-metabolizing mutant CYP102A1 M11. Because of the wide substrate selectivity of CYP102A1 M11 when compared with wild-type CYP102A1, this panel of mutants will be useful both as biocatalysts for metabolite production and as model proteins for mechanistic studies on the function of P450s in general.

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“…One obvious difference occurs at the position corresponding to Arg47 in fragment 1.1, which is located at the opening of the active site and is thought to interact with the carboxylate group of fatty acid substrates [ 29]. Substitutions of this residue in the A1 holoenzyme significantly reduce catalytic activity [ 44, 45]. In A2, the equivalent residue is Gly48, a residue that favors the binding of polycyclic aromatic hydrocarbons when present in the A1 holoenzyme [ 46].…”

Section: Resultsmentioning

confidence: 99%

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

et al. 2006

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Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ˜3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.

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Roles of key active-site residues in flavocytochrome P450 BM3

Cited by 159 publications

References 19 publications

Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase

Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase

Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

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