De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

Sheila J. Sadeghi et al

modulating the desired properties of the biosensor using protein engineering on the one hand, together with novel interfacial technologies on the other.

2. Engineering artificial redox chains

For potential applications of cytochromes P450 in biosensors it is more desirable to replace the biological electron delivery and transport system by artificial ones like electrochemical [99] or photochemical systems [111-112]. Both methods have been applied to cytochrome P450 since the early years of P450 research. Several laboratories have used various methods to reduce cytochromes P450 electrochemically [113-117]. Although some electrochemical aspects of P450s were reported more than 20 years ago [118-119], the direct, non-promoted electrochemistry of P450 is rather difficult to obtain with unmodified electrodes. The enzyme does not interact with the electrode and is denatured.

The first direct electrochemistry in solution at the edge-plane graphite electrode was reported by Hill’s group [113]. Rustling’s group has found that P450cam incorporated in lipid or polyelectrolyte film displayed the well-defined redox behaviour from its haem Fe(II/III) [ 114]. More recently, Hill’s group [115] demonstrated cyclic voltammograms on an edge-plane graphite electrode for various P450cam mutants.

The P450 enzyme of interest in the work carried out in this laboratory is P450 BM3 whose characteristics already covered in the introduction make it very interesting for biotechnological applications. However, P450 BM3 does not react with electrodes mainly due to its buried haem. The strategy adopted to tackle this problem makes use of an engineered, artificial redox chain, where electrons are conveyed to the catalytic unit via a protein known to interact with the electrode surfaces. This strategy plans to exploit the knowledge of biological ET for biotechnological purposes. In this strategy, a redox protein with well-characterised electrochemistry, flavodoxin, is used as a module to transfer electrons to the P450 unit (Figure 4).

Figure 4. Schematic representation of the proposed artificial redox chain assembly.

In order to establish the functionality of the chosen building blocks to be used for the covalent assembly of the artificial redox chains, the ET between the separate proteins

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was studied by stopped-flow spectrophotometry [120]. In the first instance the ability of flavodoxin from Desulfovibrio vulgaris (fld) to transfer electrons to simple cytochromes, cytochrome c from horse heart (hh c ) and cytochrome c553 from Desulfovibrio vulgaris (c553 ), was investigated. The knowledge gained from these systems was then applied to the more complex enzymatic system namely that of the haem domain of P450 BM3 from Bacillus meguterium (BMP) [121].

Flavodoxin (fldq) was reduced anaerobically to its semiquinone form (fldsq) in one syringe of the stopped-flow apparatus by the semiquinone radical of deazariboflavin (dRfH•) produced by photo-irradiation in the presence of EDTA (Figure 5).

Figure 5 Absorption spectra following the photoreduction of oxidisedfld under anaerobic conditions to its semrquinione form (left) and reoxidation of the latter by oxidised cytochrome followed at 580 nm by stoppedflow spectrophotometry (right)

The reaction scheme studied is summarised in the following equations, where equation [3] applies to hhc and c553 and equation [3]' applies to BMP:

Under pseudo-first order and saturating conditions, the ET process of the fld/hhc redox pair showed two components with klim of 41.45 + 4.75 s -1 and 12.15 + 2.14 s -1 and K app of 32 + 10 µM and 44 + 18 µM for the fast and slow processes, respectively. For the fld/c553

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and fld/BMP redox pairs a single component was found with a klim of0.48+_0.05 s-1 and 43.77 ± 2.18 s-1 and a Kapp of 21±6 µM and 1.23 ± 0.32 µM, respectively [89, 120].

An important factor for achieving efficient ET is the formation of an ET competent complex between the redox pairs. The effect of the electrostatic forces in producing the complexes was studied by changing the ionic strength of the protein solutions. The second-order rate constants for the reaction of fld/hh c were two orders of magnitude higher (106 M-1s-1) than the same rates measured for fld/ c553 (104 M-1S-1). Furthermore, these rates decreased monotonically with increasing ionic strength for the fld/hh c as expected for a reaction occurring between two molecules with opposite charges. The fld/ c553 and fld/BMP redox couples showed a bell-shaped behaviour due to hydrophobic as well as electrostatic interactions. The effect of solution ionic strength on the electrostatic surface potentials of all three redox couples was also calculated as shown in Figure 6. As expected, the proteins with most charged surface residues, fld and hhc, are more affected by the changes in ionic strength than c553 and BMP.

Figure 6 Surface potentials calculated for the three ET pairs fld/hhc (left), fld/c553 (centre) andfld/BMP (right) at three different solution ionic strengths, 1mM (top), 60 mM (centre) and 500 mM (right) These calculations were carried out using the program Delphi with a probe radius of l0Åfor hhc and c553 w ith I 4Åfor BMP

The ET data were analysed further using the parallel plate model developed by Tollin and co-workers [122]. This model takes into account the asymmetric distribution of charges on the surface of the protein and emphasises local electrostatic interactions between the charged moieties at the site of ET. The parameters obtained from fitting the kinetic data to this model are reported in Table 1.

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Table 1. Parameters calculatedfrom the Parallel Plate Model.

The availability of the 3D structures of these proteins allows the use of computational methods for generating a 3D model of the possible complexes. The structure of such models is important in this work for the rational design of the covalent redox chains described in the following sections. In the case of fld/hhc and fld/c553 docking simulations have been carried out [41], the results of which are generally in good agreement with the experimental data. The purported area of contact of the two proteins within both complexes show both electrostatic as well as hydrophobic interactions, with an average radius of 10 Å, well in agreement with the results obtained from the parallel plate model.

In the case of the fld/BMP complex, a model was generated by super-imposition of the 3D structure of fld on that of the truncated P450 BM3 [123]. The average radius of the contact area in this complex is 22 Å, which is considerably higher than that of the other two complexes and in good agreement with the value found from the parallel plate model. The distance between the redox centres in this complex is 18 Å, which is comparable with that found in the structure of the truncated P4.50 BM3 [123]. However, an alternative model is also possible, where the FMN region of fld is docked in the positively charged depression on the proximal BMP surface, around the haem ligand cysteine 400. This model brings the two cofactors at a closer distance of <12 Å. The two possible models may reflect the presence of dynamic events accompanying the formation and reorganisation of the ET competent complex that has also been postulated for the natural P4.50-reductase complex [SO].

The models of the ET competent complexes described above were used to generate

compartments, fld in the cytoplasm and c553 in the periplasm. A fld-c553 fusion was constructed at the DNA level, by linking the fld gene to that of c553 via a DNA sequence

codifying for a flexible seven amino acid linker (GPGPGPG). The length of this peptide linker was determined by molecular modelling experiments in which the two proteins were docked and a loop was modelled to join the C-terminus of fld to the N- terminus of c553, as shown in Figure 7. The resulting fusion gene expressed the chimeric protein of the correct molecular weight (25 kDa). Although the optical absorption spectrum of the partially purified chimeric protein showed the characteristic

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absorbencies for the oxidised haem (410 nm) and FMN (458 nm) cofactors, but the relative ratio of these cofactors was not the expected 1 : 1 possibly due to the expression of the fld-c553 protein in the cytoplasm of E. coli (the correct haem incorporation in c553 was obtained in the periplasm).

Figure 7. Modelled structure of the fld-c553 fusion protein.

The fusion of the fld-BMP system was carried out at DNA level by linking the BMP gene (residues 1-470) with that of fld (residues 1-148) through the natural loop of the reductase domain of P450 BM3 (residues 471-479). This gene fusion was achieved by ligation of the relevant DNA sequences with engineered restriction sites. A possible model of this fusion protein is shown in Figure 8.

Figure 8. Modelled structure of the fld-BMP fusion protein.

The fusion gene was correctly expressed in a single polypeptide chain. The absorption spectra of the purified chimeric protein indicated the incorporation of 1 : 1 haem and FMN. Moreover, the reduced protein was able not only to form the carbon monoxide adduct with the characteristic absorbance at 450 nm, but also to bind substrate (arachidonate) displaying the expected lowto high-spin transition from 4 19 nm to

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397 nm, indicating that this covalent complex is indeed a functional P450. The integrity of the secondary structure of the fld-BMP fusion protein was confirmed by CD spectroscopy, with a ~2% increase in the a-helix content when compared to the BMP, probably due to the addition of the engineered loop. The spectroscopic data show that the fusion protein is indeed expressed as a soluble, folded and functional protein [121].

The presence of intra-molecular ET from the domain containing the FMN to the domain containing the haem, in the presence of substrate, was studied under steadystate conditions. The flavin domain was photo-reduced by deazariboflavin in the presence of EDTA under anaerobic conditions. The subsequent ET from the flavin domain to the haem was followed by the shift of the haem absorbance from 397 nm to 450 nm in carbon monoxide saturated atmosphere.

The kinetics of the intra-molecular ET within the fld-BMP fusion protein was studied by transient absorption spectroscopy. In the experimental set up, the FMN-to- haem ET was followed by the decrease in absorbance at 580 nm of the fld sq. The ET rate measured was found to be 370 s-1. This value is comparable to that measured for the intra-protein ET from FMN to haem domain of truncated P450 BM3 (250 s-1) in which the FAD domain was removed [ 124]. These results are extremely encouraging because they demonstrate the functionality of the fld-BMP fusion protein to be equivalent to the physiological protein.

Figure 9. Figure 9. Cyclic voltammograms of' BMP and fld-BMP fusion protein in the absence/presence of neomycin on a glassy carbon electrode: BMP (I), fld-BMP (2) and fld-BMP + neomycin (3),).

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Preliminary electrochemical experiments of the fld-BMP fusion protein, free in solution were carried out using a glassy carbon electrode [125]. The cyclic voltammograms of both the fld-BMP fusion protein and BMP are shown in Figure 9. While no current was observed for P450 BM3 enzyme on the bare glassy carbon electrode, both BMP and fld-BMP show measurable redox activities. Furthermore, the fld-BMP fusion protein interacts better with the electrode as measured by the larger current compared to that of BMP. This current is further enhanced in the presence of neomycin, a positively charged aminoglycoside which is believed to overcome the electrostatic repulsion between the negatively charged fld and the negatively charged electrode surface [126].

3. Screening methods for P450 activity

After the crucial role of P450s in the determination of the pharmacological/ toxicological properties of drugs was known, the need to develop efficient systems for the in vitro screening of drug-P450 interactions emerged. Thus in the recent years a great amount of effort has been put into the generation of model systems for studying in vitro the metabolism of pharmaceutical drugs in humans. Such model systems are aiming at obtaining valid information for the metabolism of the drug candidates in vivo. Additionally, it has been widely recognised that inter-individual differences in P450 content are capable of causing significant variation in pharmacokinetics, thus leading to inaccurate estimation of the toxicological and pharmaceutical action of drug candidates. As a result, the future of pharmacokinetics seems to be getting directed towards individualised drug treatment [127], where the drug therapy to be followed will be based on the genotype of the specific patient.

On the other hand the recent development of efficient non-rational protein engineering techniques [ 1314] generating large combinatorial protein libraries has provided us with the capability to evolve protein function towards directions of choice. Strategies for screening protein libraries have been reviewed by Zhao and Arnold [128]. We tried to address both issues in our laboratory by developing a general assay for screening for turnover of compounds of interest by NAD(P)H-dependent oxidoreductases.

3.1. ASSAY METHODS FOR P450-LINKED ACTIVITY

A rapid investigation of the in vitro drug metabolism has been facilitated in the recent years by the development of high-throughput screening assays together with the advances in the creation of automated miniaturised systems for liquid handling and detection. The screens available can be categorised into

•Assays aiming at the detection of P450 inhibitors using non-specific P450 substrates which are turned over into detectable metabolites (for example fluorescent or radiolabelled) by the P450. Inhibition by a compound can thus be detected by reduction (or abolishment) of the production of the detectable metabolites [129-133].

•Assays, using an instrumental chemical analysis method, able to distinguish between the parent compound and its metabolites after turnover by the P450 [134-136].

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The last category of assays provides information on the rate of metabolism and the metabolic stability of the candidate drug, and can be very important for the early prediction of drug clearance. It can also be adapted to study drug-drug interactions. In vitro drug metabolism screening systems have been reviewed recently by Eddersaw and Dickins [135].

Apart from the assays mentioned above for screening human P450-drug interactions, a large number of assays have been proposed in the literature for screening substrate turnover by cytochromes P450 [137-139], including two assays for P450 BM3 [140-141]. Most of these screening systems are dependent on the direct or indirect detection of the product of the turnover of certain substrates by the P450 enzyme. Although such methods can be adapted for high throughput assays, their application is limited to specific P450 enzymes with particular substrates, or they could be used for activity screening of a specific cytochrome rather than for identification of substrates within a random pool of molecules. The method proposed by Sligar's group [140], presents the advantage that it can be broadly applied to any P450 enzyme. However important limitations of the method are that it is not sufficiently sensitive to allow screening pools of mutants and that it can only be applied on cell lysates, as opposed to whole cells.

3.2. DEVELOPMENT OF A NEW HIGH-THROUGH-PUT SCREENING METHOD FOR NAD(P)H LINKED ACTIVITY

Recent developments in this laboratory have led to the development of a new high- through-put screening method for NAD(P)H-linked oxidoreductase activity. This method is applicable to any enzymatic activity that uses NAD(P)H cofactors, including the catalytically self-sufficient class II, cytochrome P450 BM3. The method has been adapted to a 96-well microtiter plate-format for screening large numbers of molecules in microlitre quantities. It is based on the spectrophotometric detection of NA(D)P+ produced when a molecule of interest is being turned over by whole-cells expressing a P450 enzyme. The standard assay based on the monitoring of NADPH consumption by following the decrease of the NADPH absorbance at 340 nm could not be applied for detecting P450 activity in whole cells for the following reasons:

•light scattering by cells is interfering with the peak at 340 nm, and

•such assay would require a plate-reader able to follow enzyme kinetics. As mentioned in the introduction, assays based on expressed human P450s have actually been presented as more valid systems for the in vitro investigation of metabolic pathways in humans, since these systems can be adapted to take into account the genetic polymorphism, responsible for inter-individual differences

in metabolic profiles.

A schematic representation of the assay can be seen in Figure 10. The principal of the assay lies on the fact that the reduced and oxidised forms of NAD(P)H have a different sensitivity to destruction in extreme pH values [142-143]. Thus by changing the pH of the reaction mixture, containing the cytochrome, a substrate, the remaining NAD(P)H and the NAD(P)+ generated during catalysis, from the lower extreme of the pH scale to its other extreme, the amount of oxidised NAD(P)+ can be selectively quantified. In the

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experimental procedure, cells expressing the cytochrome of interest are aliquoted into the wells of a microtiter plate and incubated with the pool of compounds to be screened for turnover by the enzyme. The reaction is initiated by addition of NADPH. At a certain time point after the start of the reaction, the produced NADP+ is quantified: The pH is initially lowered to at least below 2.5 and subsequently risen to above 14.5. Following this procedure, NADP+ is specifically quantified by measuring the absorbance of the formed product at 360 nm. To ensure that the generated NADP' is due to coupled substrate oxidation, the possible uncoupling of reducing equivalents to the formation of hydrogen peroxide is investigated. This is measured by quantification of hydrogen peroxide using the HRP-ABTS assay [ 144].

1.Aliquots of cells expressing P450 BM3

2.+ Pool of compounds

3.+ NADPH

4.pH<2.5

4a. Uncoupling assay

5. pH >14.5 (HRP-ABTS)

6. Incubation in dark

8. Active, coupled substrates

Figure 10 Schematic representation of the screening protocol for identifying novel substrates of cytochromes P450 within a random group of compounds

Applying the assay to four known substrates of P450 BM3, the fatty acids arachidonic and lauric, the solvent 1,1,2,2-tetrachloroethane and the anionic surfactant sodium dodecyl sulphate, a 360 nm signal of only 3% was given by cells in the absence of substrate, compared to cells in the presence of substrate, under the optimum experimental conditions. Control experiments with cells non-transformed, or transformed with the same vector expressing other redox enzymes not able to turn over

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the substrates of interest in the presence of NADPH, were also run. They all demonstrated that the assay is specifically detecting P450 BM3 oxidising activity in whole-cells.

The proposed method presents the advantage that it can be generalised for the screening of substrates or inhibitors of any P450 enzyme, or any oxidoreductase in general, utilising NADPH or NADH (both have same behaviour in extreme basic/acidic conditions) as donor or acceptor of reducing equivalents. At the same time it can be applied for the screening of variants within a library of random mutants of a NADPHdependent reductase for the specificity of interest.

In the work carried out in this laboratory, the viability of this new assay has been put to the test by screening

•a series of furazan derivatives, potential pharmaceuticals, against the wild type

P450 BM3,

•a series of random mutants of P450 BM3 against target pollutants,

•an engineered, catalytically self-sufficient, chimeric bacterial-human P450 enzyme against some known substrates of both proteins.

3.3. VALIDITY OF THE NEW SCREENING METHOD

The validity of the assay has been demonstrated by investigating the interaction of the heterologously expressed in E. coli bacterial cytochrome P450 BM3 with a group of 1,2,5-oxadiazole 2-oxide (furoxan) derivatives, potential pharmaceuticals for the treatment of cardiovascular diseases, and their 1,2,5-oxadiazole (furazan) analogues (Figure 11) [145].

Figure 11. Structures of the ten furazan derivatives screened for turnover by wild-type cytochrome P450 BM3. The derivatives marked with + were found to cause increased consumption of NADPH, relative to the background, in contrast to analogues indicated by -, which showed NADPH-oxidising activity at background levels.

Within a group of ten analogues screened for interaction with the wild type P450 BM3, seven were identified as positives. The results from the assay in whole-cells were confirmed for all analogues, by following NADPH consumption by the purified

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