Biocatalytic Hydroxylations Catalyzed by Cytochromes P-450—Problems and Prospects

Due to their selectivity biocatalytic hydroxylations catalysed by monooxygenases or dioxygenases arouse considerable interest. The most studied and distributed type of monooxygenases are cytochromes P-450, which are a supergene family of proteins that catalyse the oxidation of lipophilic compounds through the insertion of 1 oxygen atom of O₂ into the substrate. For biocatalytic processes those cytochromes deserve attention, are involved in the catabolism of nutrients in microorganisms or in the metabolism of physiological substrates in higher eucaryotes. Mammalian cytochromes P-450, which catalyse the oxidation of xenobiotics (synthetic drugs, pesticides, hazardous waste compounds, are hardly suitable for this purpose. Processes with immobilized cytochromes P-450 can be excluded. The use of wild or genetically transformed strains of microorganisms is a suitable way.     

Cytochromes c and P-450 as terminal acceptors in a reconstituted system of mitochondrial hydroxylation

Both cytochromes c and P-450 can be reduced in vitro by a mitochondrial hydroxylation chain consisting of flavoprotein, adrenodoxin and NADPH as electron donors. The effects of pH, ionic strength, content and stoichiometry of intermediate electron carriers on the reduction rate of cytochromes c and P-450 have been compared. The data obtained demonstrate that the reduction of these cytochromes proceeds by different ways. It has been found that cytochrome c has an inhibitory effect on cytochrome P-450 reduction. On the other hand, cytochrome P-450 remarkably activates the process of cytochrome c reduction.     

Cytochromes P450 in gibberellin biosynthesis

The gibberellins (GAs) are an important class of plant growth regulators that are active in many aspects of plant growth and development. GAs are synthesized by a complex pathway involving three enzyme classes spanning different subcellular compartments. One of these enzyme classes is the cytochrome P450s which catalyze a number of oxidation steps in the middle part of the pathway. Mutants in these cytochrome P450-mediated steps in a number of species have been crucial in isolating the genes encoding these enzymes and have also played an important role in understanding GA physiology. GAs are also synthesized by fungi, in a biosynthesis pathway largely catalyzed by cytochrome P450s. The fungal pathway appears to have evolved independently to that of higher plants.

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Cytochromes P450 and drug resistance

Cytochromes P450 are the key enzymes for activating and inactivating many drugs, in particular anticancer drugs. Therefore, individual expression levels of cytochromes P450 may play a crucial role in drug safety and drug efficacy. Overexpression of cytochrome P450 may yield rapid turnover and elimination of drugs before the target site was reached and any pharmacological effect is observed. Therefore, it may be vital to know the individual cytochrome P450 status in order to select the appropriate drug before drug resistance occurs. Expression levels and activity of cytochromes P450 depend on many different factors. These factors include tissue and organ specific expression, sex- and age-dependent expression, genetic differences yielding polymorphic forms, competitive inhibition or induction of cytochromes P450 due to multiple drug interaction, nutrition and diet. Genetically engineered test cells defined for cytochromes P450 are available for studying drugs for metabolic activation and for identifying the metabolically competent cytochrome P450 isoform.     

Cytochromes P450 and drug discovery

Cytochromes P450 (CYP) are a superfamily of haem-containing proteins distributed widely throughout nature. Historically, they have a central role in drug metabolism and following the advent of genomics they have been shown to have key roles in the biosynthesis of natural products which are used as medicines. Herein, we provide an overview of CYP systems with particular emphasis on their role as drug targets, their involvement in drug biosynthesis and potential strategies for developing new derivatives of drugs based on CYP engineering. The applied importance of CYPs for medicinal and biotechnological applications will also be discussed.     

Cytochromes P450 as versatile biocatalysts

Cytochromes P450 are ubiquitously distributed enzymes, which were discovered about 50 years ago and which possess high complexity and display a broad field of activity. They are hemoproteins encoded by a superfamily of genes converting a broad variety of substrates and catalysing a variety of interesting chemical reactions. This enzyme family is involved in the biotransformation of drugs, the bioconversion of xenobiotics, the metabolism of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins, bile acids, the conversion of alkanes, terpenes, and aromatic compounds as well as the degradation of herbicides and insecticides. There is also a broad versatility of reactions catalysed by cytochromes P450 such as carbon hydroxylation, heteroatom oxygenation, dealkylation, epoxidation, aromatic hydroxylation, reduction, dehalogenation (Sono, M., Roach, M.P., Coulter, E.D., Dawson, J.H., 1996. Heme-containing oxygenases. Chem. Rev. 96, 2841-2888), (Werck-Reichhart, D., Feyereisen, R., 2000. Cytochromes P450: a success story. Genome Biol. 1 (REVIEWS3003)), (Bernhardt, R., 2004. Cytochrome P-450. Encyclopedia Biol. Chem. 1, 544-549), (Bernhardt, R., 2004. Optimized chimeragenesis; creating diverse P450 functions. Chem. Biol. 11, 287-288), (Guengerich, F.P., 2004. Cytochrome P450: what have we learned and what are the future issues? Drug Metab. Rev. 36, 159-197). More than 5000 different P450 genes have been cloned up to date (for details see: ). Members of the same gene family are defined as usually having > or =40% sequence identity to a P450 protein from any other family. Mammalian sequences within the same subfamily are always >55% identical. The numbers of individual P450 enzymes in different species differ significantly, showing the highest numbers observed so far in plants. The structure-function relationships of cytochromes P450 are far from being well understood and their catalytic power has so far hardly been used for biotechnological processes. Nevertheless, the set of interesting reactions being catalysed by these systems and the availability of new genetic engineering techniques allowing to heterologously express them and to improve and change their activity, stability and selectivity as well as the increasing interest of the industry in life sciences makes them promising candidates for biotechnological application in the future.     

Cytochromes P450 in phenolic metabolism

Three independent cytochrome P450 enzyme families catalyze the three rate-limiting hydroxylation steps in the phenylpropanoid pathway leading to the biosynthesis of lignin and numerous other phenolic compounds in plants. Their characterization at the molecular and enzymatic level has revealed an unexpected complexity of phenolic metabolism as the major route involves shikimate/quinate esters and alcohol/aldehyde intermediates. Engineering expression of CYP73s (encoding cinnamate 4-hydroxylase), CYP98s (encoding 4-coumaroylshikimate 3′-hydroxylase) or CYP84s (encoding coniferaldehyde 5-hydroxylase) leads to modified lignin and seed phenolic composition. In particular CYP73s and CYP98s also play essential roles in plant growth and development, while CYP84 constitutes a check-point for the synthesis of syringyl lignin and sinapate esters. Although recent data shed new light on the main path for lignin synthesis, they also raised new questions. Mutants and engineered plants revealed the existence of (an) alternative pathway(s), which most likely involve(s) different precursors and oxygenases. On the other hand, phylogenetic analysis of plant genomes show the existence of P450 gene duplications in each family, which may have led to the acquisition of novel or additional physiological functions in planta. In addition to the main lignin pathway, P450s contribute to the biosynthesis of many bioactive phenolic derivatives, with potential applications in medicine and plant defense, including lignans, phenylethanoids, benzoic acids, xanthones or quinoid compounds. A very small proportion of these P450s have been characterized so far, and rarely at a molecular level. The possible involvement of P450s in salicylic acid is discussed.     

Cytochromes P450: a success story

Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.     

Cytochromes P450 for engineering herbicide tolerance

In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.     

Cytochromes P450 and insecticide resistance.

The cytochrome P450-dependent monooxygenases (monooxygenases) are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Monooxygenase-mediated metabolism is a common mechanism by which insects become resistant to insecticides as evidenced by the numerous insect species and insecticides affected. This review begins by presenting background information about P450s, the role of monooxygenases in insects, and the different techniques that have been used to isolate individual insect P450s. Next, insecticide resistance is briefly described, and then historical information about monooxygenase-mediated insecticide resistance is reviewed. For any case of monooxygenase-mediated resistance, identification of the P450(s) involved, out of the dozens that are present in an insect, has proven very challenging. Therefore, the next section of the review focuses on the minimal criteria for establishing that a P450 is involved in resistance. This is followed by a comprehensive examination of the literature concerning the individual P450s that have been isolated from insecticide resistant strains. In each case, the history of the strain and the evidence for monooxygenase-mediated resistance are reviewed. The isolation and characterization of the P450(s) from the strain are then described, and the evidence of whether or not the isolated P450(s) is involved in resistance is summarized. The remainder of the review summarizes our current knowledge of the molecular basis of monooxygenase-mediated resistance and the implications for the future. The importance of these studies for development of effective insecticide resistance management strategies is discussed.     

Cytochromes P450: Roles in Diseases

The cytochrome P450 superfamily consists of a large number of heme-containing monooxygenases. Many human P450s metabolize drugs used to treat human diseases. Others are necessary for synthesis of endogenous compounds essential for human physiology. In some instances, alterations in specific P450s affect the biological processes that they mediate and lead to a disease. In this minireview, we describe medically significant human P450s (from families 2, 4, 7, 11, 17, 19, 21, 24, 27, 46, and 51) and the diseases associated with these P450s.     

Specificity of Isatin Interaction with Cytochromes P450

Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this study, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase systems: five isoforms of cytochromes P450, two isoforms of cytochrome b₅, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown high affinity specific binding of isatin only to cytochromes P450 (the equilibrium dissociation constant (K_d) is about 10^–8 M).     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Bacterial cytochromes P-450

The cytochromes P-450 (P-450s) constitute an extremely large family ('superfamily') of haemoproteins that catalyse the oxidation of a wide range of physiological and non-physiological compounds. A remarkable feature of the P-450s is the manipulation of the same basic structure and chemistry to achieve an enormous range of functions in organisms as diverse as bacteria and man. Indeed, the P-450s have been described as ‘the most versatile biological catalyst known’. Much research is focussed on mammalian P-450s, with their roles in such processes as steroid transformations and the metabolism of carcinogens and other xenobiotics. However, our knowledge of the structure and function of the P-450s has been advanced by analysis of a limited number of its bacterial members, primarily P-450cam from Pseudomonas putida. Four P-450 structures have been solved to date, all of which are from bacterial sources. The aim of this review is to assess current knowledge of the many bacterial P-450s, with emphasis on their diverse biological roles and on the advances in our knowledge of this extremely important enzyme class, which have been made feasible through their study.     

Insect cytochrome P-450

Summary Two approaches may be used to study the function of cytochrome P-450 in insects: (a) an evaluation of the spectral and catalytic properties of the hemoprotein while associated with microsomal membranes; (b) the solubilization, resolution and purification of the microsomal mixed-function oxidase system. The first approach has provided some understanding of the biochemical factors involved in the metabolism of a variety of compounds, including pesticides, drugs, hormones and many other xenobiotics. However, solubilization of the monooxygenase system allows the study of each of its components individually, providing a better insight on the sequence of events leading to the hydroxylation of a substrate, the type of intermediates formed, and the rate-limiting step(s). This report discusses studies carried out with the monooxygenase system associated with microsomal membranes, as well as procedures to solubilize and partially purify its components from housefly microsomes. The latter involves solubilization with either Triton X-100 or sodium cholate, followed by either ammonium sulfate fractionation, Sephadex G-200, DEAE-Sephadex A-50 column chromatography or by-amino-n-octyl-Sepharose 4B affinity chromatography. These procedures have shown that two cytochrome P-450 species (P-450 and P-450_I) are present in microsomes isolated from a resistant housefly strain. Induction with either naphthalene or phenobarbital appears to increase cytochrome P-450_I preferentially.An invited article.