Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

Plant glycosyltransferases: their potential as novel biocatalysts

The potential application of glycosyltransferases in glycoconjugate synthesis has attracted considerable interest from the biotechnology community in recent years. This concept article focuses on the current understanding of the chemistry of a family of plant enzymes capable of glycosylating small lipophilic molecules. These enzymes are discussed in terms of their regio- and enantioselective substrate recognition, sugar-donor selectivity and their utility as biocatalysts in whole-cell systems.     

Electrochemistry of P450cin: new insights into P450 electron transfer

Electrochemistry of bacterial cytochrome P450cin (CYP176A) reveals that, unusually, substrate binding does not affect the heme redox potential, although a marked pH dependence is consistent with a coupled single electron/single proton transfer reaction in the range 6 < pH < 10.     

Mechanisms of reaction in cytochrome P450: Hydroxylation of camphor in P450cam

The fundamental nature of reactivity in cytochrome P450 enzymes is currently controversial. Modelling of bacterial P450cam has suggested an important role for the haem propionates in the catalysis, though this finding has been questioned. Understanding the mechanisms of this enzyme family is important both in terms of basic biochemistry and potentially in the prediction of drug metabolism. We have modelled the hydroxylation of camphor by P450cam, using combined quantum mechanics/molecular mechanics (QM/MM) methods. A set of reaction pathways in the enzyme was determined. We were able to pinpoint the source of the discrepancies in the previous results. We show that when a correct ionization state is assigned to Asp297, no spin density appears on the haem propionates and the protein structure in this region remains preserved. These results indicate that the haem propionates are not involved in catalysis.     

An evaluation of molecular models of the cytochrome P450 Streptomyces griseolus enzymes P450SU1 and P450SU2

Summary P450SU1 and P450SU2 are herbicide-inducible bacterial cytochrome P450 enzymes from Streptomyces griseolus. They have two of the highest sequence identities to camphor hydroxylase (P450cam from Pseudomonas putida), the cytochrome P450 with the first known crystal structure. We have built several models of these two proteins to investigate the variability in the structures that can occur from using different modeling protocols. We looked at variability due to alignment methods, backbone loop conformations and refinement methods. We have constructed two models for each protein using two alignment algorithms, and then an additional model using an identical alignment but different loop conformations for both buried and surface loops. The alignments used to build the models were created using the Needleman-Wunsch method, adapted for multiple sequences, and a manual method that utilized both a dotmatrix search matrix and the Needleman-Wunsch method. After constructing the initial models, several energy minimization methods were used to explore the variability in the final models caused by the choice of minimization techniques. Features of cytochrome P450cam and the cytochrome P450 superfamily, such as the ferredoxin binding site, the heme binding site and the substrate binding site were used to evaluate the validity of the models. Although the final structures were very similar between the models with different alignments, active-site residues were found to be dependent on the conformations of buried loops and early stages of energy minimization. We show which regions of the active site are the most dependent on the particular methods used, and which parts of the structures seem to be independent of the methods.     

Iron Porphyrin as a Cytochrome P450 Model for the Degradation of Dye

Organic dyes are widely used in the textile, biological, medical and other fields. However, a serious environmental problem has appeared because of the presence of organic dyes in industrial aqueous effluents. Thus, the efficient treatment of organic dyes in industrial wastewaters is currently in real demand. The current study investigated the oxidative degradation of the organic dye gentian violet by meso-tetra(carboxyphenyl) porphyriniron(III), [Fe^III(TCPP)] as a cytochrome P450 model and iodosylbenzene (PhIO) as an oxidant at room temperature. The degradation reaction was monitored by UV–vis absorption spectroscopy via the observation of UV–vis spectral changes of the gentian violet. The results showed that the efficiency of catalyzed degradation reached more than 90% in 1 h, indicating the remarkable oxidative degradation capacity of the [Fe^III(TCPP)]/PhIO system, which provided an efficient approach for the treatment of dyeing wastewater.     

Cyclopropyl containing fatty acids as mechanistic probes for cytochromes P450

[reaction: see text] The mechanism of aliphatic hydroxylation by cytochromes P450 has been the subject of intense debate with several proposed mechanistic alternatives. Various cyclopropyl containing compounds (radical clocks), which can produce both unrearranged and ring opened products upon oxidation, have been key tools in these investigations. In this study, we introduce several cyclopropyl containing fatty acids 1a-4a with which to probe the mechanism of P450s capable of fatty acid hydroxylation. The probes are shown to be capable of distinguishing radical from cationic intermediates due to the rapid equilibration of isomeric cyclopropyl cations. Ring opening of a radical intermediate in an oxidative transformation is expected to yield a single rearranged alcohol, whereas a cation isomerizes prior to ring opening, leading to two isomeric homoallylic alcohols. Oxidation of these probes by P450(BM3) and P450(BioI) gives results consistent with a radical but not a cationic intermediate in fatty acid hydroxylation by these enzymes. Quantitation of the unrearranged and ring opened products gives remarkably homogeneous rates for oxygen rebound of (2-3) x 10(10) s(-1). The effects of introduction of a cyclopropane ring into a fatty acid upon the regiochemistry of hydroxylation are discussed.     

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF‐7 breast cancer cells are treated with CYP‐polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site‐specific CYP activity in tumor tissues are discussed.

     

Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3

The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.     

Drug Oxidation by Cytochrome P450BM3: Metabolite Synthesis and Discovering New P450 Reaction Types

There is intense interest in late‐stage catalytic C?H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450_BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α‐hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α‐functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late‐stage C?H activation catalysts.     

P450 versus P420: correlation between cyclic voltammetry and visible absorption spectroscopy of the immobilized heme domain of cytochrome P450 BM3

Cyclic voltabsorptometry is used for the first time to distinguish and characterize electrochemically the active (P450) and inactive (P420) forms of cytochromes P450 immobilized on an electrode during voltammetry experiments. This was achieved by using the heme domain (BMP) of the bacterial cytochrome P450 BM3 from Bacillus megaterium (CYP102A1) immobilized on mesopouros tin-oxide (SnO2) electrodes. We demonstrate that the formation of either the P450 form or the P420 one can be obtained by modifying the mesoporous electrode surface with polycations with different properties such as polyethylenimmine (PEI) and polydiallyldimethylammonium chloride (PDDA). Potential step spectroelectrochemistry allowed measurement of reduction potentials of the active P450 form. Values of -0.39+/-0.01 V and -0.58+/-0.01 V (both versus Ag/AgCl) were calculated for the active P450 form immobilized on the BMP/PDDA-SnO2 and BMP/PEI-SnO2 electrodes, respectively. The cyclic voltabsorptometric experiments showed how, when both the active and inactive forms are present on the PEI film, the inactive P420 species tends to dominate the cyclic voltammetric signal.     

Kinetic isotope effects implicate the iron-oxene as the sole oxidant in P450-catalyzed N-dealkylation

Multiple oxidants have been implicated as playing a role in cytochrome P450-mediated oxidations. Herein, we report results on N-dealkylation, one of the most facile reactions mediated by P450 enzymes. We have employed the N-oxides of a series of para-substituted 13C2H2-labeled N,N-dimethylanilines to function as both substrates and surrogate oxygen atom donors for P450cam and P4502E1. Kinetic isotope effect profiles obtained using the N-oxide system were found to closely match the profiles produced using the complete NAD(P)H/NAD(P)-P450 reductase/O2 system. The results are consistent with oxidation occurring solely through an iron-oxene species.     

High throughput screening and QSAR-3D/CoMFA: useful tools to design predictive models of substrate specificity for biocatalysts

After a hierarchical microbial screening process, new microorganisms have been discovered that act as biocatalysts for the stereoselective oxidation of secondary alcohols or for ketone reduction. Oxidation activity is more widespread in yeasts and bacteria, while actinomycetes, filamentous fungi and yeasts present the highest reduction activities. QSAR-3D/CoMFA is an adequate technique to design predictive models of the biocatalysts' activity. In this paper CoMFA models are designed to compare the activities of the biocatalysts selected for the oxidation of alcohols and for the reduction of ketones, starting from the results obtained during the screening process. These models are useful for learning about the activity of these microorganisms and to compare the substrate specificity requirements between alcohol oxidation and ketone reduction biocatalysts.     

Cytochrome P450s: creating novel ligand sets

Cytochromes P450 are a ubiquitous group of hemoproteins that perform vital cellular reactions in all lifeforms. Until recently, it was thought that P450s contained non-covalently bound heme. However, it was established that covalent linkage of the heme macrocycle occurs naturally in one major group of the P450 superfamily. The reaction involves heme linkage to a conserved amino acid and is autocatalytic, occurring as a consequence of P450 turnover. This finding presents opportunities to engineer biotechnologically important P450s to covalently link the heme, in order to stabilize cofactor binding and to enhance operational stability of these P450s. This opportunity has been taken in studies on two important bacterial P450s and has produced variants with intriguingly different properties. In this article we survey the developments in the field, the relationships with heme macrocycle ligations in other proteins and the important impact that recent studies of heme ligation have had on our general appreciation of P450 structure and mechanism.     

A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.     

Methoxy-resorufin ether as an electrochemically active biological probe for cytochrome P450 O-demethylation

This paper describes the utilisation of methoxy-resorufin ether as an electrochemical probe for studying cytochrome P450 CYP6G1. Methoxy-resorufin ether is well established as a versatile substrate for cytochrome P450, as its demethylated product, resorufin, is a fluorophore. We show that in addition to these established properties, methoxy-resorufin ether also exhibits reversible two electron transfer on glassy carbon and edge plane graphite electrodes. Cyclic voltammetry measurements and differential pulse voltammetry measurements show that methoxy-resorufin ether can be easily detected at low concentrations (down to 200 nM) in a conventional three electrode electrochemical cell. These properties of methoxy-resorufin ether mean that it could be used as an electrochemical probe, to follow the rate of its demethylation by CYP6G1. We show that electrochemical measurements could discriminate between the enzyme activity of protein microsomes taken from two strains of Drosophila melanogaster (fruit fly).