Improving catalytic properties of P450 BM3 haem domain electrodes by molecular Lego

Fantuzzi, Andrea; Meharenna, Yergalem T.; Briscoe, Paul B.; Sassone, C.; Borgia, Beatrice; Gilardi, Gianfranco

doi:10.1039/b517472d

Cited by 25 publications

(22 citation statements)

References 17 publications

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“…4. This phenomenon has also been observed with other P450 isoenzymes such as CYP2B4 (Shumyantseva et al, 2004), CYP2E1 (Fantuzzi et al, 2004), P450 BM3 (Fantuzzi et al, 2006), CYP2C9 (Johnson et al, 2005), and CYP3A4 (Joseph et al, 2003).…”

Section: Characterization Of Cyp2c9-immobilized Gold Electrodesupporting

confidence: 66%

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Yang¹,

Kabulski²,

Wollenberg³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Cytochrome P450 (P450) enzymes typically require the presence of at least cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of xenobiotics. To address whether the need for redox transfer proteins and the NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer (SAM) through which a current could be applied. Cyclic voltammetry demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the electrode and fast electron transfer between the heme iron and the gold electrode. To determine whether this system could metabolize warfarin analogous to microsomal or expressed enzyme systems containing CYP2C9, warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled potential was applied.The expected 7-hydroxywarfarin metabolite was observed, analogous to expressed CYP2C9 systems, wherein this is the predominant metabolite. Current-concentration data generated with increasing concentrations of warfarin were used to determine the MichaelisMenten constant (K m ) for the hydroxylation of warfarin (3 M), which is in good agreement with previous literature regarding K m values for this reaction. In summary, the CYP2C9-SAM-gold electrode system was able to carry out the metabolism of warfarin only after application of an electrical potential, but in the absence of either CPR or NADPH. Furthermore, this system may provide a unique platform for both studying P450 enzyme electrochemistry and as a bioreactor to produce metabolites without the need for expensive redox transfer proteins and cofactors.Cytochrome P450 (P450) enzymes are important for drug metabolism in humans, accounting for ϳ75% metabolism of drugs (Williams et al., 2004). Numerous factors affect the P450 metabolism rate and the resulting metabolite structure including electron transfer, protein-protein interactions, concentration and structure of the substrate, and the source and specific means whereby the P450 was prepared (Guengerich, 2007). In practice, it is difficult to isolate the individual contributions of these factors because they are often interdependent. Thus, it is of interest to devise model platforms that can be used to independently control these parameters to monitor their respective effects on metabolism.P450 catalysis requires a constant supply of NADPH as the electron source and cytochrome P450 reductase (CPR) to deliver the electrons.In attempts to obviate the requirement of these redox partners/cofactors for catalysis, P450 enzymes have been immobilized on electrodes so that the electrode supplies electrons to drive the P450 catalytic cycle (Estabrook et al., 1996;Reipa et al., 1997;Gilardi et al., 2002) with effective electrical communication between the electrode and the enzyme being critical (Yang et al., 2008). Direct adsorption of enzymes on bare electrodes, such as gold, platinum, and graphite, results in diminished biocatalytic activity (Habermüller et al., 2000;Joseph et al., 2003;Shumy...

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Section: Characterization Of Cyp2c9-immobilized Gold Electrodesupporting

confidence: 66%

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Yang¹,

Kabulski²,

Wollenberg³

et al. 2009

Drug Metab Dispos

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“…Despite the full bacterial enzyme or only the heme domain (BMP) have been immobilized on different electrode surfaces [ 117–119], only few reports have shown that the protein is able to turn over substrates by using the reducing equivalents donated by the electrode surface. Our group achieved catalysis on the substrate p -nitrophenol with the BMP immobilized on modified glassy carbon electrodes [ 120] and the chimeric protein BMP/Fld, where the redox protein flavodoxin from Desulfovibrio vulgaris was fused to BMP, resulted in a 6-fold increase of the amount of the reaction product p -nitrocatechol [ 120]. One key factor in electrochemically-driven P450 catalysis firstly reported by our group [ 121, 122], is that strategies for immobilisation of electrode surfaces leading to high electron transfer values do not necessarily lead to catalytically active enzymes.…”

Section: Optimization Of P450 Bm3 As a Biocatalyst For Drug Metabomentioning

confidence: 99%

Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

Nardo

Gilardi

2012

IJMS

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Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported.

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“…However, the use of polymer or a polyelectrolyte enhances the preservation of the native structure, thereby increasing electron transfer between the enzyme and the electrode. Recently, a molecular Lego approach (assembling non-physiological redox modules, generating artificial redox chains, which leads to a new multi-domain construct) has been developed to improve the electrochemical and catalytic properties of CYP102 [63]. …”

Section: Applications Of Cytochrome P450 Biocatalystsmentioning

confidence: 99%

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Kumar

2010

Expert Opinion on Drug Metabolism & Toxicology

144

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Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts.

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Improving catalytic properties of P450 BM3 haem domain electrodes by molecular Lego

Abstract: In this work the catalytic properties of a cytochrome P450 immobilised onto an electrode surface are improved by means of the molecular Lego approach.

Cited by 25 publications

References 17 publications

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

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