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CHAPTER 1 GENERAL INTRODUCTION Chapter 1 General Introduction Introduction 1. Role of drug metabolism in drug development 4 1.1. Metabolites in safety testing (MIST) 1.2. Enzyme involved in drug metabolism 1.3. Biosynthesis of reactive metabolites 1.4. Biological screening for active metabolites 2. Microbial conversion of steroid compounds 12 3. Cytochrome P450s 15 3.1. Human P450s 3.2. Structural organization of P450s 3.3. The catalytic cycle 4. Cytochrome P450 BM3 20 4.1. Engineering Cytochrome P450 BM3 4.2. P450 BM3 in biocatalysis 4.3. Key residues in the active site of P450 BM3 5. Spin lattice relaxation NMR 27 6. Scope and objective of this thesis 29 7. Outline of this thesis 30 Chapter 1 General Introduction 1. Role of drug metabolism in drug discovery and development Most xenobiotic substances such as drugs undergo an array of biotransformation reactions after internal exposure to humans. Drug metabolism by the host system is one of the most important determinants of the pharmacokinetic profile of a drug. Biotransformation of drugs can have different effects, such as the formation of chemically stable metabolites, which are devoid of pharmacological or toxicological activities, or the generation of short lived chemically reactive metabolites, which can lead to toxicological side effects (1). Furthermore, drug metabolism can also lead to formation of pharmacologically active metabolites which might contribute significantly to drug action in vivo, or may be responsible for pharmacologically-based adverse drug reactions. A relatively novel approach in modern drug development is the use of pharmacologically active metabolites as potential resources for drug discovery and development. There are several advantages for screening drug candidates for active metabolites during drug discovery. For example drug metabolites can show superior properties compared to the lead itself, such as improved pharmacodynamics, improved pharmacokinetics, lower probability of drug-drug interactions, less variable pharmacokinetics and/or pharmacodynamics, improved overall safety profile and improved physicochemical properties (e.g. solubility) (1). Examples of active metabolites of previously marketed drugs that have been developed into drugs are listed in Table 1. Table 1. Pharmacologically active metabolites developed as new drugs (2) Parent drugs Metabolite drugs Biotransformation Brand name Allopurinol Oxypurinol Oxidation of xanthine Oxyprim Amitriptyline Nortryptyline N-Demethylation Aventyl Bromhexine Ambroxol N-Demethylation & Mucosovan Hydroxylation Diazepam Oxazepam N-demethylation & Serax Hydroxylation Etretinate Acitretin Deesterification Soriatane Hydroxyzine Cetirizine Carboxylation Zyrtec Imipramine Desimipramine N-Demethylation Norpramin Loratadine Desloratadine Descarboethoxylation Clarinex Loxapine Amoxapine N-Demethylation Asendin Phenacetin Acetaminophen O-Deethylation Tylenol Terfenadine Fexofenadine Carboxylation Allegra Thioridazine Mesoridazine S-Oxidation Serentil Chapter 1 General Introduction For example, fexofenadine, the primary metabolite of terfenadine was developed as antihistamine agent with selective peripheral histamine H1-receptor antagonist activity. Fexofenadine is a safer drug than terfenadine because it does not has the ability to cause cardiotoxicity by hERG inhibition. In some instances, metabolic transformation can also produce reactive or toxic intermediates or metabolites, with potential toxicological implications (3). Hence, a good understanding of the metabolism of a new chemical entity is needed early in the drug discovery process. 1.1. Metabolites in safety testing (MIST) Drug metabolism and pharmacokinetics are crucial factors for successful drug development. Inappropriate pharmacokinetics previously was one of the main reasons of attrition in drug development. Although inappropriate pharmacokinetics has become a less important reason for attrition, less progress has been made in decreasing the attrition due to drug toxicity. The preparative synthesis of drug metabolites is currently of primary importance in industry in order to assess potential toxicity, drug-drug interactions and to examine metabolic pathways (4). In fact, testing the toxicities and biological activities of human metabolites of drugs is important in drug development to assure effectiveness and safety (5). The importance of metabolite identification and quantification is stressed by the guidelines published in 2008 by the US food and drug administration (FDA). The so-called Metabolites in Safety Testing (MIST) guidelines describe the studies recommended to perform on metabolites having more than 10% systemic exposure of the parent drug to support human safety. A decision diagram of this is depicted in Figure 1. These guidelines challenged the development of biocatalytic systems for the generation of large amounts of drug metabolites as well as analytical technologies dealing with their quantification and identification of drug metabolites in an efficient and intelligent way (7). Chapter 1 General Introduction Figure 1. Decision tree flow diagram of the MIST guidelines as determined by the FDA, based on (6). 1.2. Enzymes involved in drug metabolism Drug metabolism is typically divided in two phases. Phase I metabolism includes many types of functionalization reactions, e.g. oxidation, reduction, hydrolysis, hydration and dehalogenation reactions (9). Cytochrome P450s represent the most important class of enzymes involved in phase I metabolism, being involved in 75-80% of metabolism of marketed drugs, Figure 2. Other phase I enzymes include monoamine oxidases, flavincontaining oxygenases, amidases and esterases (9). Phase II metabolism involves conjugation of polar groups (e.g. glucuronic acid, sulfate, and amino acids) to drugs and/or phase I drug metabolites to further increase their hydrophilicity and facilitate excretion. These biotransformation reactions involve glucuronidation, sulfation, GSH conjugation, acetylation, amino acid conjugation and methylation reaction (10). Phase II enzymes include UDP-glucuronosyltransferase (UGTs), sulfotransferases (STs), N-acetyl transferases (NATs), methyl transferases and glutathione S-transferases (GSTs) (10), Figure 2. Almost all phase I and phase II enzymes can catalyze the formation of stable metabolites, as well as chemically reactive intermediates that may lead to the generation of adverse drug reactions (11). Chapter 1 General Introduction Figure 2. Phase I and phase II metabolism of currently marketed drugs (adapted from (8)). The percentage of phase I and phase II metabolism of drugs that each enzyme contributes is estimated by the relative size of each section of the corresponding chart. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; DPD, dihydropyrimidine dehydrogenase; NQO, NADPH quinine oxidoreductase. COMT, catechol Omethyltransferase; GST, glutathione S-transferase; HMT, histamine methyltransferase; NAT, N-acetyltransferase; TPMT, thiopurine methyltransferase; UGTs, uridine 5’triphosphate glucuronosyltransferases. 1.3. Role of reactive metabolites in adverse drug reactions Adverse drug reactions (ADRs), as defined by the World Health Organization, are noxious, unintended and undesirable effects of a drug, which occurs at therapeutical doses used in humans and which require hospitalization of patients to recover (11). Although much effort has been spent to the development of safer drugs, ADRs still constitute a major reason for attrition in drug development and withdrawal or restriction of marketed products. Therefore, prevention of ADRs constitutes a major challenge for the pharmaceutical industry (3). Approximate 75% of ADR in patients are classified as Type A ADR which involve lifethreatening exaggerated pharmacological activity (on-target and/or off-target) due to unanticipated increased plasma concentrations of the parent drug. This class of ADR often results from drug-drug interactions or genetic deficiency of an enzyme which is involved in the major pathway of metabolism. For the other classes of ADR formation of reactive drug metabolites are considered to play a crucial role (12). The main difficulty for pharmaceutical industry is the fact that ADRs cannot always be predicted in preclinical animal studies. In particular idiosyncratic drug reactions (IDRs; Type B ADR) are difficult to predict as they normally have a very low incidence, have a Chapter 1 General Introduction delayed time of onset, do not necessarily show classic dose-response relationships and are not predictable from the known pharmacology of the drug. No predictive animal model is currently available and IDRs therefore remain poorly understood. Figure 3 shows a general scheme depicting the role of reactive metabolites in the development of toxicity. Drugs bioactivation can be catalyzed by both phase I and phase II enzymes. Different types of reactive metabolites can be distinguished such as electrophiles, radicals and reactive oxygen species. Electrophilic metabolites can react with nucleophilic sites in proteins and/or DNA. Free radicals possess unpaired electrons and can abstract hydrogen atoms from polyunsaturated fatty acids in membranes which will initiate the destructive process of lipid peroxidation, and can disrupt the cellular redox-state leading to oxidative stress and subsequent toxicity (12). Figure 3. Scheme depicting the role of drug metabolism in determining the toxic outcome in ADRs. Table 2 gives examples of drugs whose toxicities are attributed to covalent binding of electrophilic metabolites to proteins. Bioactivation of xenobiotics which contain certain functional groups such as tertiary amine, furan ring or acetylene group, can also cause mechanism-based inactivation of P450, leading to adverse clinical drug-drug interactions (13-14). Chapter 1 General Introduction Table 2. Examples of drugs and reactive metabolites possibly involved in ADRs (12-15). Drugs Reactive intermediate Toxicity Acetaminophen Quinone-imine Hepatotoxicity Carbamazepine 2-Hydroxy, Quinone-imine Agranulocytosis, Aplastic anemia Clarithromycin Nitroalkane Hypersensitivity Clozapine Nitrenium ion Agranulocytosis Dapsone Hydroxulamine, Nitroso Hemolysis, hypersensitivity Diclofenac Acylglucuronide, Benzoquinone imine Hepatotoxicity Halothane Trifluoroacetyl Hepatitis Indomethacin Iminoquinone Hypersensitivity, Agranulocytosis, Hepatotoxicity Isonizaid Isonicotinic acid, acetylating species Hypersensitivity Phenacetin p-Nitrosophenetole Hepatotoxicity Procainamide Hydroxylamine, Nitroso Agranulocytosis Tacrine 7-OH-tacrine Hepatotoxicity Tamoxifen N-oxide, N-oxide-epoxide Carcinogenesis Ticlodipine Keto, S-oxide Agranulocytosis, aplastic anemia Tielinic acid Thiopene S-oxide Hypersensitivity, hepatitis Troglitazone Conjugates, Benzoquinone, Quinone Hepatotoxicity epoxide Valproic acid Acyl glucuronides Hypersensitivity, Hepatotoxicity Detection of reactive metabolites is difficult due to the chemical reactivity of the shortlived intermediates and to the usually low amounts present in incubations. The most common way to screen for formation of reactive metabolites is to trap them with model nucleophiles such as GSH and to analyze the formed adducts with spectroscopic techniques. The endogenous tripeptide GSH can trap different types of RIs including quinones, quinoneimines, iminoquinone, methides, epoxides, areneoxides and nitrenium ions (15). The corresponding GSH adducts are typically analyzed by liquid-chromatography mass spectrometry (LC-MS). However, a major disadvantage is that structural information of the adducts obtained by LC-MS/MS experiments is limited, as it may be insufficient in identifying the exact position of oxidation, to differentiate stereoisomers, or to provide the exact structure of the metabolite (16). Complementing MS data with Nuclear Magnetic Resonance (NMR) experiments is required for complete structural elucidation of drug metabolites and/or resulting adducts. A limitation of NMR, however, is its relative insensitivity, as it requires several milligrams of pure metabolite. Chapter 1 General Introduction The generation of pure metabolites can be achieved by organic synthesis, electrochemical oxidation of the parent drug or by biocatalysis using appropriate enzymes. The synthesis by organic chemistry is often complicated by the lack of appropriate synthetic routes for specific metabolites and may yield only low amounts of the desired product that has to be purified subsequently. Electrochemical oxidation has been used to generate drug metabolites and has the advantage that it can be easily scaled up. However, with this technique not all the relevant enzymatic metabolites can be formed. Electrochemistry has been previously used to generate GSH adducts of clozapine (17) and troglitazone (18). Biocatalysis by human or bacterial P450s can also be used for the scaling up of metabolite production (19). For example, human relevant diclofenac metabolites were synthesized by microbial fermentation in large amount (up to 170 mg) and their structures were elucidated by MS and NMR (20). More recently the use of highly active bacterial P450 BM3 mutants for the generation of reactive metabolites of drugs has been explored. Damsten et al. showed that BM3 mutants could be used for the generation of human relevant reactive metabolites of clozapine, acetominophen, diclofenac (21), and trimethoprim (22). Boerma et al. showed that P450 BM3 mutants with high capacity to activate drugs (clozapine, acetaminophen and troglitazone) into relevant reactive metabolites can be employed to produce protein adducts to study the nucleophilic selectivity of highly reactive electrophiles (23). Dragovic et al. applied highly active BM3 mutant as bioactivation system to study the role of human GSTs in the inactivation of reactive intermediates of clozapine (24). 1.4. Biological screening for active metabolites To assess the biological activity of drug metabolites there are several approaches available. The traditional approach is to isolate and purify all the metabolites from in vitro incubations with subcellular fractions such as liver microsomes or intact cellular systems (e.g. epatocytes) containing a full complement of drug metabolizing enzymes. An alternative approach is to use bioassay-guided methods where biological samples containing biotransformation products are first evaluated for their pharmacological activity prior to isolation of the metabolites. The bioassay methods may be based on the assessment of the pharmacological activity using in vitro ligand binding (25, 26), cell based assays (1), or in vivo pharmacological assays (1). One of the major bottlenecks in in vitro assays is the lack of possibility to screen affinity or activity of individual compounds in complex mixtures. In general, two approaches can be used to measure the effect of individual compounds in a mixture. One strategy is to combine HPLC with fractionation techniques to enable the compounds in the mixture to be separated and allow screening of the individual components using plate reader assays. Chapter 1 General Introduction The other available strategy is the on-line high resolution screening technique (HRS) depicted in Figure 4 (27-29). HRS enables the screening of individual compounds in complex mixtures by coupling a separation technology, usually gradient HPLC, to post-column biochemical detection assays on-line. This technique has already been successfully used to develop a number of antibody- (30) and biotin- (31), receptor- (32), enzyme- (33) (34) (35), and MS-based (36) screening assays. The HRS platforms are especially attractive when complex mixtures such as metabolic incubations (37), combinatorial mixtures (38), or natural extracts (39) are screened. In contrast to conventional high-throughput screening (HTS), isolation of individual metabolites not showing affinity can be avoided. Thus, the laborious development of high-yield synthesis and isolation methods can be limited to compounds with the desired affinity. Figure 4. Scheme of the online setup. The system enables the separation of mixtures and subsequent parallel detection of enzyme binding and accurate mass. The samples are injected into and separated by an HPLC system. The eluent is split between HR-MS and enzyme binding detection. The fraction which enters the enzyme binding detection is mixed with the target (e.g. p38) and the tracer and incubated after each mixing step, 24 s for the target–ligand interaction and 11 s for the target–tracer interaction. Finally, the fluorescence is measured as readout of affinity towards the target. In parallel, the second part of the eluent is analyzed by HR-MS delivering structural information to identify the small molecules tested. Adapted from (28). 2. Microbial conversion of steroid compounds Chapter 1 General Introduction Totally, about 300 approved steroid drugs are known to date, and this number tends to grow (40). Steroid pharmaceuticals are ranked among the most marketed pharmaceuticals and represent the second largest category next to antibiotics. The research on steroid drugs started in 1950, with the discovery of the pharmacological effects of cortisol and progesterone, two endogenous steroids, and with the identification of the 11-hydroxylation activity of a Rhizopus species (41-42). Microbial steroid transformation is a powerful tool for generation of novel steroidal drugs, as well as for efficient production of steroid-containing active pharmaceutical ingredients (APIs) and key intermediates. Steroid hydroxylation is a key tool to develop steroid drugs with improved potency, longer half-lives in the blood stream, simpler delivery methods and reduced side effects (43). The therapeutic area of hydroxy-steroids is very wide: they are used as anti inflammatory, immunosuppressive, progestational, diuretic, anabolic and contraceptive agents (41, 4447). Applications are found in the treatment of adrenal insufficiencies, in the prevention of coronary heart disease, as anti-fungal agents, as anti-obesity agents, and in the inhibition of HIV integrase, the prevention and treatment of infection by HIV and in the treatment of declared AIDS (43). The complex structure of the steroid molecule requires complicated, multi-step schemes for the chemical synthesis of steroid compounds. The selective oxidation of an unactivated, aliphatic C-H bond to an alcohol is a challenging problem in synthesis (48). Chemical synthesis requires the use of reagents as pyridine, sulfur trioxide or selenium dioxide, whose disposal constitutes an environment issue (43). Moreover, the chemical catalysts used often show very low regioselectivity. Steroid hydroxylation by microorganisms represents a powerful alternative to chemical synthesis. Current trends in microbial hydroxylation studies cover the search for novel biocatalysts capable of performing reactions targeting specific positions in the steroid molecule that are particularly important for the pharmaceutical industry (7α, 9α, 11α, 11β, 16α, 17α), production of novel hydroxysteroids with potent therapeutic properties, and the construction of novel biocatalysts by genetic engineering (40). Examples are given in Table 3. Cytochrome P450s play a very important role in endogenous steroid metabolism. Testosterone hydroxylation in particular has been studied extensively with human liver microsomal P450s. For example, CYP3A4, the most abundant P450 in human liver and small intestine, catalyzes the monohydroxylation at 10 different positions of testosterone (8). Hence testosterone hydroxylation patterns have been utilized as probes for the presence and characterization of P450s (49) (50). Hydroxylation of testosterone at 1-, 2-, 2-, 6-, 6-, 7-, 7-, 15-, 15-, 16-, 16and 17-positions has been reported in human and rat liver microsomes (Figure 5). Table 3. Examples of steroid hydroxylation in microrganisms Substrate Product Microrganism Ref. Chapter 1 7-Hydroxylation Testosterone Pregnenolone Epiandrosterone 7-Hydroxulation DHEA 7-15Dihydroxylation DHEA General Introduction 7-OH-testosterone 7-OH-pregnenolone 7-OH-epiandrostenone Botrytis cinerea AM235 Fusarium oxysporum var.cubense Mortierella isabellina AM212 (49) 7- and 7-derivatives 7-OH DHEA M.racemosus Botryodiplodia malorum CBS 134.50 (51) (52) 7-15-diOH-DHEA (3,7,15- C.lini CBS 112.21 triOH-androst-5-ene-17-one) 7-15Dihydroxylation Corynespora cassiicola CBS 161.60 17-OH-progesterone 8-OH-derivative, along with 15-hydroxy derivative 9-hydroxylation Testosterone 9-OH-AD, 9-OH-testosterone R.equi ATCC 14887 Progesterone 9-OH-progesterone Expression of 9-hydroxylase from M.smegmatis in E.coli BL21 Androsterone 9-OH-adrenosterone, 9-OH- Cunninghamella elegans TSY-0865 11-keto-testosterone 11-Hydroxylation Testosterone Rhizopus stolonifer 11-OH-testosterone ATCC 10404, Fusarium lini NRRL 68751 11-hydroxylation Progesterone T.harzianum, T.hamatum 11-OH-progesterone 14-Hydroxylation Testosterone A.wentii MRC 200316 14-OH-testosterone 15-Hydroxylation Testosterone F.oxysporum var. cubense 15-OH testosterone Progesterone 15-OH progesterone 15-Hydroxylation Testosterone Solvent tolerant 15-OH-testosterone Pseudomonas putida S12 expressing B.megaterium steroid hydroxyase CYP106A2 16-Hydroxylation Testosterone 2,16,17-triOH-androst-4-en- Whetzelinia sclerotiorum ATCC 18687 3-one 17-Hydroxylation Progesterone B.sphaericus ATCC 245, 17-OH-progesterone B.sphaericus ATCC 7063, B.sphaericus ATCC 13805 (50) (52) (53) (54) (55) (56) (57) (58) (59) (60) (61) (62) (63) Chapter 1 General Introduction Figure 5. Hydroxylation of Testosterone by human P450s (adapted from (51)) Figure 6. Testosterone hydroxylation by bacterial P450s (adapted from (40)). Chapter 1 General Introduction Hydroxylation of Testosterone by bacterial P450s has also been extensively studied (Figure 6) (51). Profiling this hydroxylation provides useful information for characterizing bacterial P450s as monogenic traits and for comparing bacterial P450s with human P450s. The features and functionality of steroid hydoxylases have been recently reviewed by, e.g. Bernhardt (2006) (52), Hannemann et al (2007) (53), Novikova et al. (2009) (54) and Kristan and Lanisnik-Rizner (2012) (55). 3. Cytochrome P450s Cytochrome P450s (P450s or CYPs) constitute a family of monoxygenases involved in the biotransformation of drugs, xenobiotics, alkanes, terpenes, and aromatic compounds, in the metabolism of chemical carcinogens, the biosynthesis of physiologically relevant compounds as steroids, fatty acids, eicosanoids, fat-soluble vitamins, bile acids and in the degradation of pesticides (52). They are able to catalyze a number of difficult oxidative reactions, such as C-H bond hydroxylation, N-dealkylation, N-hydroxylation, O-dealkylation, S-oxidation and epoxidation of numerous endogenous and exogenous compounds (3). An overview of the different reactions catalyzed by P450s is listed in Table 3. The broad substrate acceptance and this huge diversity in catalyzed reactions make the class of P450 very promising biocatalysts in industrial processes. In fact, in recent years there has been an increasing interest in the application of CYP biocatalysts for the industrial synthesis of bulk chemicals, pharmaceuticals, agrochemicals, and food ingredients, especially when a high grade of stereo and regioselectivity is required (57). Moreover P450s are of great interest in drug metabolism. They are ubiquitous enzymes often involved in the oxidation of drugs resulting in the generation of metabolites that are more easily excreted, thus modulating the toxicity of these compounds (57). 3.1. Human P450s The human genome encodes for 57 functional P450s (58) of which approximately a dozen are involved in drug metabolism. The P450s mostly involved in metabolism of drugs in humans are listed in Table 5, where the main reaction and substrate specificities are indicated. Genetic polymorphisms that may alter the enzyme activity have been reported for a number of P450s (59). While for the P450s 1A2, 2A6, 2B6 and 2C8 the clinical effects due to these polymorphisms are minor, major effects are observed for 2C9, 2C19, and 2D6 (60). Polymorphisms in these isoenzymes affect metabolism of 20% to 30% of the clinically used drugs and this has become a major issue in the discovery and development of drugs and other NCEs (61). Table 4. List of different reactions catalyzed by P450s (56) Reaction type Example Chapter 1 General Introduction Aliphatic hydroxylation Aromatic hydroxylation Epoxidation Heteroatom dealkylation Alkyne oxygenation Heteroatom oxygenation Aromatic epoxidation and NIH-shift Dehalogenation Dehydrogenation Reduction Cleavage of esters Table 5.Substrate specificity of human CYPs CYP CYP1A2 Substrate properties Drug/Substrates Planar, polyaromatic compounds Clozapine Phenacetine Olanzepine Imipramine Propranolol Theophylline Reaction Oxidation O-deethylation Oxidation Demethylation 4-hydroxylation Chapter 1 3.2. Structural organization of P450s General Introduction Chapter 1 General Introduction All P450s are heme-containing proteins containing an iron atom at the center of the heme which is also complexed by a cysteine residue as axial ligand. Based on their redox partner, P450s can be classified in different classes (Figure 7): Figure 7. Structural organization of P450s: (a) Class I (mostly bacterial P450s), (b) Class II (mostly microsomal P450s) and (c) P450 BM3. (Adapted from (62)) Class I P450s (a) require two redox proteins: a small redox 2Fe-2S iron-sulfur ferredoxin and a FAD or FMN containing reductase. Mitochondrial P450s and most soluble bacterial P450s belong to this class (63). In class II (b), electrons are transferred via only one reductase having a FAD and a FMN domain. Microsomal P450s that are attached to the endoplasmatic reticulum belong to this class (63). Most bacterial P450s belong to class I. However, there are exceptions: in 1981 Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium was identified. P450 BM3 (c) is soluble and uses a Class II redox system. This soluble bacterial enzyme contains the P450 monoxygenase domain fused to the electron transfer flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD) reductase domain in a single polypeptide (63). In contrast, mammalian CYPs require additional redox partners as cytochrome P450 reductase (CPR) and often cytocrome b5 and lipids (64, 65). 3.3. The catalytic cycle The general catalytic cycle for substrate hydroxylation by P450s is depicted in Figure 8. Chapter 1 General Introduction Figure 8. The catalytic cycle of P450s, adapted from (66) In the resting state, in absence of substrate, the ferric iron atom is six-coordinated, complexed with a water molecule (I). Binding of a substrate displaces the water molecule (II). This causes the ferric spin state to convert from low spin to high spin, a transition that can be monitored spectrophotometrically since the Soret band undergoes a “type I” shift from ca. 418 nm to ca. 390 nm. A single electron supplied by a reduced pyridine nucleotide (NADPH or NADH) reduces the heme iron to the ferrous state (III). Subsequently, dioxygen binds to the ferrous iron generating the oxy-complex (IV). A second electron is then transferred, creating a ferric peroxy complex (V). Protonation of the terminal oxygen atom gives “Compound 0”, a hydroxyperoxy adduct (VI). Addition of a second hydrogen atom followed by loss of water gives “Compound I” (VII), a ferryl species thought to be the active entity in most P450 oxidations, which has been characterized recently (67). This complex abstracts hydrogen from the substrate (VIII) after which the hydroxyl group formed rebounds to the substrate radical, resulting in formation of the hydroxylated product which releases from the active site. NAD(P)H consumption is not necessarily fully coupled to product formation (68). If dioxygen binding is hindered, the second electron transfer will be too slow, resulting in the loss of superoxide from (IV) (superoxide uncoupling). If substrate fitting in the active site cannot prevent water encroachment, (VI) will be protonated at the iron-bound oxygen, resulting in the loss of Chapter 1 General Introduction hydrogen peroxide (peroxide uncoupling). If a substrate binds with no hydrogen atom conveniently positioned for abstraction, the oxygen atom in (VII) could be reduced to water (oxidase uncoupling). Those alternative pathways can hamper the use of P450s in biocatalysis because the expensive electron donating cofactor is used in futile redox cycling instead of product formation. Moreover the peroxide and superoxide formed by uncoupling can damage the biocatalyst. 4. Bacterial Cytochrome P450 BM3 Bacterial P450 systems often exhibit much higher catalytic activities than membranebound eukariotic P450s and are easy to handle in the laboratory due to their solubility and high expression level in heterologous hosts (69). In particular Cytochrome P450 BM3 (BM3) is considered as one of the most promising monoxygenases for biotechnological applications as it possesses the highest activity ever recorded for a P450 (63, 70, 71) and can be easily expressed at high yield in Escherichia coli. The natural substrates of BM3 are C12-C18 fatty acids which are hydroxylated at very high activity at subterminal positions (72). However, over the recent years, several research groups have succeeded in expanding the substrate selectivity of P450 BM3 and improving its catalytic properties by site-directed and/or random mutagenesis. Through rational redesign and directed evolution, BM3 mutants have been obtained that are able to oxidize aromatics (73), alkanes (74), hydrocarbons (75), carboxylic acids (76) and pharmaceuticals (21, 22, 77-84). Recently a large body of research has been conducted to broaden and alter the substrate specificity of bacterial CYPs in an effort to generate “human like” P450 activities (57). The demand of “humanized” bacterial P450 with high activity is constantly growing to meet the needs of the fine-chemical synthesis, pharmaceutical production and bioremediation industries (85). 4.1. Engineering P450 BM3 The application of engineered bacterial P450s also have application in pharmacology and toxicology as it enable the generation of substantial amounts of drug metabolites as well as in optimization of lead compounds (86). Figure 9 shows examples for substrates of engineered cytochrome P450 BM3 variants. In order to improve the selectivity of oxidation to a specific product, the substrate from which it is formed must be constrained to bind in a particular orientation (87). This can be obtained by rational redesign of the active site, for example introducing polar residues or bulky side-chains (88-90). Chapter 1 General Introduction Figure 9. Examples for substrates of engineered cytochrome P450 BM3 variants. (Adapted from (62)). Directed evolution, in which mutations are introduced at random positions using errorprone PCR, is a valuable complementary tool to site-directed mutagenesis. This approach has the advantage of being able to generate unpredictable mutations with unanticipated beneficial properties. The benefits of the two strategies can be combined using rational evolution, in which selected residues are subjected to random or semi-random mutagenesis. Alternatively, directed evolution can be used to identify sites suitable for targeting, followed by site-saturation mutagenesis to establish which mutations are most effective in those positions. For example, Dietrich et al. (91) developed a novel semi biosynthetic route for the production of artemisinin, a highly important antimalarian agent. By rational redesign of Chapter 1 General Introduction the active site they introduced mutations F87A/A328L in the active site of substrate promiscuous P450 BM3: mutation F87A appeared to relieve the steric hindrance imposed upon the substrate amorphadiene and allowed an increased access to the heme group, while mutation A328L decreased the mobility of amorphadiene in the active site, promoting epoxidation. A similar approach has been used by Seifert et al. (75), who focused on the same hotspots F87 and A328, creating a minimal and highly enriched P450 BM3 mutant library, by mutating those two positions to all possible combinations of five hydrophobic aminoacids. In this way, by systematically altering the size of the side chains in those two positions, a broad range of binding site shapes was generated to convert a range of differently sized and shaped terpenes (75). Recently, Seifert et al. (92) set up an iterative approach comprising successive rounds of modeling, designing of focused mutant libraries and screening, to identify a mutant for the production of the highly valuable product perillyl alcohol. It is widely accepted that increased regio-, stereoselectivity is the result of a reduced number of substrate orientations close to the heme, as discussed in chapter 4 of the present thesis (82, 90, 93). Considerable effort has been invested for improving the coupling efficiency (ratio of product formed to NAD(P)H cofactor consumed) of P450 BM3 by rational mutagenesis. However the efficiencies obtained are still far lower than with native enzyme-substrate. Detailed information on the structural determinants of coupling with novel substrates are missing, therefore it is difficult to rationalize mutations to improve coupling efficiencies. However, cost-effective, high-throughput screening methods will need to be developed to assess relative coupling efficiency among P450s in mutant libraries (86). Properties that need to be improved to enable the application of P450s in industrial processes are: TTN (total turnover number), substrate acceptance, regio-and stereoselectivity, activity (kcat, KM), inhibition and coupling efficiency (70). Also improvement of physical properties as thermostability, solvent tolerance, oxidative stability and substrate and product tolerance should be addressed (94). 4.2. P450 BM3 in biocatalysis One emerging application of P450s for the pharmaceutical industry is the generation of large amount of drug metabolites, as discussed above (86). Several groups showed drug metabolite preparation using human P450s expressed in Escherichia coli and in insect cells (95). The Arnold group showed that human metabolites of propranolol could be produced by mutants of bacterial P450 BM3 heme domain in a reaction driven by hydrogen peroxide (5). Yields obtained were comparable to those produced by recombinant human P450s in bacterial or baculovirus bioreactors (96). Recently, Dubey et al. set up a biotechnological production of anticancer drug (colchicine derivatives) using P450 BM3 as biocatalyst obtaining a yield of 7.3 g/L in 70 L fermentor (97). Kim at al. applied BM3 wt Chapter 1 General Introduction and a set of mutants for the generation of the human metabolite piceatannol from the anticancer-preventive agent resveratrol (77), for the generation of human chiral metabolites of simvastatin and lovastatin (79) and for the generation of human metabolites of 7-ethoxycoumarin (78). The Arnold group compiled a panel of 120 mutants to prepare nearly all human metabolites and a number of novel hydroxylated derivatives for the drugs verapamil and astemizole (81). This panel of enzymes could also be applied for the generation of new chemical entities (NCEs) to diversify lead compounds. Within any catalyst panel both extremes of regioselectivity can be useful: enzymes selectively producing individual metabolites and less selective enzymes to survey metabolite possibilities (81). A recent area of interest is the design and development of novel P450s as biocatalysts of reactions of commercial interest, such as in pharmaceutical or fine chemical synthesis (86) as well as for the optimization of lead compounds. An example of the application of BM3 mutants in chemical synthesis is the chemoenzymatic elaboration of monosaccharides applied by the Arnold group (98). Regioselective deprotection of monosaccharide substrates using engineered P450 BM3 demethylases provides a highly efficient method to obtain valuable intermediates that can be converted to a wide range of substituted monosaccharides and polysaccharides (98). Engineered Cytochrome P450 BM3s were also applied for the enantioselective hydroxylation of 2-arylacetic acid derivatives and could produce an authentic human metabolite of buspirone with high activity and selectivity (80). Rentmeister et al. showed that products hydroxylated by BM3 can be subsequently fluorinated in a chemo-enzymatic process that greatly simplifies the insertion of fluorine into these structures at specific positions (99). Fluorination has become an important tool in drug discovery and development as it can modulate the pharmacokinetic and the pharmacological properties of drugs and lead compounds. In particular, fluorination can improve membrane permeability, metabolic stability and/or receptor-binding properties of bioactive molecules (99). 4.3. Key residues in the P450 BM3 active site Control of regio- and stereoselectivity of biocatalysts is one of the major challenges in biotechnology, as a limited number of residues in the substrate pocket appear to possess significant selectivity-influencing powers. Ala82 and Phe87 are among those. In Figure 10 key residues of the substrate channel and the active site of substrate-bound P450 BM3 are shown. P450 BM3 crystallizes in a “precatalytic conformation” in which the substrate is located too far to the heme iron for the oxidation to take place (87). Therefore it is unclear whether the residues located in the substrate access channel are also catalytically relevant. Also the spin relaxation NMR experiments of reduced P450 BM3 suggested that the substrates are 6 Å closer to the porphyrin ring than in the oxidized form, indicating that structural rearrangements occur in order to allow the substrate to Chapter 1 General Introduction move closer to the heme iron (100). In the present thesis mutagenesis studies at position 87 (101) (84) and position 82 (89) will be presented. Figure 10. The substrate access channel (A) and active site (B) of NPG-bound P450BM3 (Adapted from (66)) Phe 87. Phe 87 is one of the most studied residues of the active site of BM3 and it is object of chapter 2 and 3 of the present thesis. In the substrate-free crystal structure, this residue lies perpendicular to the porphyrin system, but in the substrate-bound crystal, the Chapter 1 General Introduction orientation of the aromatic ring changes to parallel to the plane of the heme (102) (103) (63). It has been proposed that this motion influences the orientation of the substrate relative to the catalytic center and it is responsible for the displacement of the axial water ligand from its coordination site (104). Phe87 forms with Phe81 a small hydrophobic pocket within the active site that sequesters the -end of fatty acids. It seems likely that small hydrophobic substrates could preferentially bind in the pocket generating non-productive complexes (105). Phe87 is the most commonly mutated residue of P450 BM3 (Table 6). Site-directed mutagenesis studies proved that Phe87 plays a key role in the control of reaction selectivity (106) (107). Typically, Phe87 was mutated to aminoacids with less bulky side chains, in order to destroy the hydrophobic pocket, creating more space close to the heme, to accomodate larger substrates (73-75, 77, 80, 103, 105, 108-116). Such substitutions, however, lead to a decreased coupling efficiency of NADPH consumption to product formation due to a less efficient exclusion of water from the active site (105). The F87V mutation is a common component of several drug metabolizing variants (69) (117). Moreover, it has been shown that mutation F87V significantly affected the stereoselectivity of arachidonic acid epoxidation (106) and the activity towards indoles (118). Li et al. also showed that mutation F87V is beneficial for oxidation of polycyclic aromatic hydrocarbons, such as naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene (119). In the present thesis saturation mutagenesis was applied to evaluate the effect of all possible amino acids in this position on the metabolism of alkoxyresorufins, testosterone and clozapine (84, 101). Ala 82. The existence of a hydrophobic pocket between Phe81 and Phe87 clearly affects the attempts to engineer BM3 towards novel target substrates. Huang et al. (90) were the first to try to “fill” the hydrophobic pocket rather than destroying it, by mutating Ala82 to large hydrophobic residues like Phe and Trp. The mutants A82F and A82W showed a remarkable increase in affinity (800-fold) for fatty acids. Moreover, the efficiency of indole hydroxylation increased, due both to increased Kcat/KM values and to increased coupling efficiency, achieved by a more efficient exclusion of water from the active site. Mutation at this position will be object of chapter 4 of the present thesis. Table 6. Reported mutations at position Phe87: ALA (29 )(73-75) ( 81) (98, 99) (107 )(120-134) ARG (101, 135) ASN (101) ASP (101) Chapter 1 CYS GLN GLU GLY HIS ILE LEU LYS MET PRO SER THR TRP TYR VAL SS General Introduction (101, 133) (101, 135) (101, 135) (5, 74, 81, 98, 101, 122, 130, 135-143) (101, 135) (75, 81, 92, 98, 99, 135, 144, 145) (75, 81, 92, 121, 124, 135, 144, 145) (101, 135) (101, 135) (101, 135) (101, 135) (101, 135) (101, 135) (101) (106) (135) (136) (140) (146-147) (69,74, 75, 81, 82, 98, 99, 106, 118, 119, 142, 144, 145,148-153) (5, 91, 101, 118, 144, 145, 154, 155) Table 7. Reported mutations at position Ala82: CYS (81, 135, 144) GLY (81, 99, 144, 155) ILE (81, 90, 144, 156) LEU (5, 80, 81, 99, 121, 134, 144, 156, 157) PHE (81, 90, 144, 156, 158) PRO (5, 81) SER (81, 144, 155) THR (81, 144) TRP (90, 99, 135, 159, 160) VAL (156) SS (5, 144, 155) 5. Spin lattice relaxation NMR (or T1 paramagnetic relaxation NMR) In order to understand the structural basis that governs the regioselectivity of the metabolism of a certain substrate or the specificity of a given P450 for a particular drug, it 1 is important to determine the structure of the substrate binding sites. For that purpose H NMR appears as a powerful and attractive technique, since measurements of Chapter 1 General Introduction paramagnetic relaxation can be used to determine the distances between the heme iron and protons of the bound substrate in P450-substrate complexes (161). The heme iron atom of a CYP is paramagnetic when in oxidized state. Hydrogen atoms in a magnetic field will re-align in the direction of this field after their orientation has been flipped with a radio-frequency pulse. The velocity of this re-alignment (relaxation rate) is dependent on the local strength of the magnetic field in the direct proximity of the hydrogen atom. So a hydrogen atom close to the heme in the active site of a CYP, will relax faster due to the magnetic moment of the heme iron atom. The relationship between the iron atom to hydrogen atom distance and the rate of relaxation has been established by Solomon and Bloembergen (162) and can be used to measure substrate orientations in CYP active sites. A disadvantage of the technique is that high concentrations (~mM) of ligands need to be present to measure NMR spectra. Furthermore, average substrate active site orientations do not always match the metabolic profile (163). An overview of reported spin lattice relaxation studies using CYPs is given in Table 6. In chapter 4 of the present thesis this technique has been successfully applied to determine the orientation of testosterone in two BM3 mutants (M11 and M11 A82W) to rationalize the different regioselectivity obtained in the metabolic profile. Table 8. Spin lattice relaxation NMR studies used to determine active site ligand orientations in CYPs. CYP 102 Ligand 12-Br-lauric acid 1A Paracetamol 1A2 Caffeine 2B Paracetamol Distances (A)* 7.8 (C1H)-16.3 (C12H) 5.9 (phenylH)6.7(methylH) 6.5 (N1methylH)6.7 (N3 and N7 methylH) 5.8 (methylH)- 6.3 (phenylH) Major reaction C2-OH Benzoquinone formation Reference (163) N1-demethylation (163) Inactive (164) (164) Chapter 1 General Introduction *the minimal and maximal hydrogen atoms to heme iron atom measured are given. **Measured in the F483I mutant of CYP2D6, the wild type is inactive towards testosterone. 6. Scope and objectives The research presented in this thesis was part of a wider interdisciplinary project “Metabolic stability assessment as a new tool in the Hit-to-Lead selection process and the generation of new lead compound libraries” financed by the Dutch Top-Institute Pharma consortium (grant D2-102). Several industrial (Merck/MSD (Oss, NL) and QPS (Groeningen, NL)) and academic (Vrije Universiteit Amsterdam and Radboud Universiteit Chapter 1 General Introduction Nijmegen) partners participated in this project that led to several publications and four PhD theses. The aim of the project was the application of new, more efficient and selective P450 BM3 mutants for the generation of biotransformation products, being new chemical starting points for lead optimization (lead libraries), followed by the determination of their binding affinity (e.g. competitive fluorescence detection assay) and chemical structural characterization (LC-MS/MS-NMR) in a hyphenated approach. The general objectives of the present thesis are: Engineering drug metabolizing P450 BM3 mutants mimicking human P450s and mutants with unique catalytic properties based on regio- and stereoselective metabolism of diagnostic substrates. Utilization of “humanized” drug metabolizing P450 BM3 mutants for large scale production of physiologically relevant human metabolites of lead compounds. Figure 11 shows the general strategy applied in this research: by site-directed, random or saturation mutagenesis BM3 mutants are engineered to obtain highly active mutants able to metabolize drugs and drug-like molecules. The metabolic mixtures obtained by incubation of diagnostic substrates with engineered BM3 mutants are then screened to verify the acquisition of certain properties: HRS enables the selection of mutants able to produce metabolites that show pharmacological activity, GSH trapping enables the identification of mutants able to produce reactive metabolites (subsequently trapped by GSH), screening by UPLC allows the identification of changes in regio- and stereoselectivity in a quick and informative way. Selected mutants that acquired a certain property (e.g. production of high amounts of a pharmacologically active metabolite, high selectivity for the production of reactive metabolites, high selectivity of the production of a certain hydroxy-steroid) are then applied as biocatalysts for the large scale metabolite production in order to enable pharmacological and toxicological evaluation of drug metabolites and for their structural elucidation by NMR. Chapter 1 General Introduction Figure 11. General strategy applied for the research presented in this thesis, leading to a “metabolite production and profiling platform” 7. Outline of this thesis The work presented in this thesis shows application of BM3 mutants as biocatalyst for three purposes: For the regio- and stereo- selective hydroxylation of lead compounds (e.g. steroids) For the generation of reactive metabolites For the generation of pharmacologically active metabolites / lead optimization Regio- and stereo-selective hydroxylation of unactivated C-H bonds of natural or synthetic compounds is one of the biggest challenges in synthetic organic chemistry. In particular steroid hydroxylation is extremely challenging due to the complexity of the steroid molecule that requires complicated, multi-step schemes involving protecting groups and subsequent regeneration. In chapter 2 and 4 of the present thesis cytochrome P450 BM3 mutants are applied as biocatalysts for the regioselective hydroxylation of testosterone and norethisterone. In chapter 2 a site saturation mutagenesis library of BM3 mutants was applied to evaluate the effect of a single mutation in the active site of BM3 on the regioselective hydroxylation of testosterone and on the coupling efficiency in alkoxyresorufin oxidation. In chapter 4, a single active site substitution was applied in order to reduce the substrate mobility of testosterone, thus improving the regioselectivity Chapter 1 General Introduction of hydroxylation. Spin lattice relaxation NMR was applied to determine how this single mutation affected the orientation of the substrate within the active site of two BM3 mutants, M11 and M11 A82W. The generation of reactive metabolites is important from a toxicological point of view to be able to identify potentially toxic compounds that may be involved in ADRs or IDRs. Chapter 3 of the present thesis focuses on the application of P450 BM3 mutants for the generation of reactive metabolites of clozapine, which is a drug known to be responsible of severe ADRs. By screening of a minimal library of BM3 mutants, a mutant was selected for the generation of large amounts of all major human relevant GSHconjugates of clozapine to enable their structural elucidation by NMR. The generation of pharmacologically active metabolites is important from a “lead optimization” point of view. Sometimes drug metabolites possess improved pharmacological activity and improved physico-chemical properties, compared to the lead itself. In chapter 5 of the present thesis a focused library of BM3 mutants has been applied for the generation of human relevant bioactive metabolites of p38 kinase inhibitor Tak-715. The HRS screening applied enabled the identification and pharmacological characterization of the drug metabolites in a quick and informative online mode. Large scale incubation with BM3 mutants and isolation of active metabolites by Prep-LC allowed their structural elucidation by NMR. The last part of this thesis, chapter 6 concerns an overall summary of the work described in the thesis including general conclusions and perspectives for future work. References Chapter 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. General Introduction Fura, A., Shu, Y. Z., Zhu, M., Hanson, R. L., Roongta, V., and Humphreys, W. G. (2004) Discovering drugs through biological transformation: role of pharmacologically active metabolites in drug discovery, J Med Chem 47, 43394351. Kang, M. J., Song, W. H., Shim, B. H., Oh, S. Y., Lee, H. Y., Chung, E. Y., Sohn, Y., and Lee, J. (2010) Pharmacologically active metabolites of currently marketed drugs: potential resources for new drug discovery and development, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 130, 1325-1337. Kumar, S. Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation, Expert Opin Drug Metab Toxicol 6, 115-131. Schroer, K., Kittelmann, M., and Lutz, S. Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis, Biotechnol Bioeng 106, 699-706. Otey, C. R., Bandara, G., Lalonde, J., Takahashi, K., and Arnold, F. H. (2006) Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450, Biotechnol Bioeng 93, 494-499. www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm079266.pdf. (2008). Nedderman, A. N. (2009) Metabolites in safety testing: metabolite identification strategies in discovery and development, Biopharmaceutics & drug disposition 30, 153-162. Evans, W. E., and Relling, M. V. (1999) Pharmacogenomics: translating functional genomics into rational therapeutics, Science 286, 487-491. Woolf, T. F., and Jordan, R. A. (1987) Basic concepts in drug metabolism: Part I, Journal of clinical pharmacology 27, 15-17. Jordan, R. A., and Woolf, T. F. (1987) Basic concepts in drug metabolism: Part II, Journal of clinical pharmacology 27, 87-90. Gomes, E. R., and Demoly, P. (2005) Epidemiology of hypersensitivity drug reactions, Current opinion in allergy and clinical immunology 5, 309-316. Williams, D. P., Kitteringham, N. R., Naisbitt, D. J., Pirmohamed, M., Smith, D. A., and Park, B. K. (2002) Are chemically reactive metabolites responsible for adverse reactions to drugs?, Current drug metabolism 3, 351-366. Fontana, E., Dansette, P. M., and Poli, S. M. (2005) Cytochrome p450 enzymes mechanism based inhibitors: common sub-structures and reactivity, Current drug metabolism 6, 413-454. Zhou, S., Chan, E., Lim, L. Y., Boelsterli, U. A., Li, S. C., Wang, J., Zhang, Q., Huang, M., and Xu, A. (2004) Therapeutic drugs that behave as mechanismbased inhibitors of cytochrome P450 3A4, Current drug metabolism 5, 415-442. Caldwell, G. W., and Yan, Z. (2006) Screening for reactive intermediates and toxicity assessment in drug discovery, Current opinion in drug discovery & development 9, 47-60. Prakash, C., Shaffer, C. L., and Nedderman, A. (2007) Analytical strategies for identifying drug metabolites, Mass spectrometry reviews 26, 340-369. DiMagno, E. P. (2007) The pancreatic cyst incidentaloma: management consensus?, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association 5, 797-798. Chapter 1 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. General Introduction Madsen, K. G., Gronberg, G., Skonberg, C., Jurva, U., Hansen, S. H., and Olsen, J. (2008) Electrochemical oxidation of troglitazone: identification and characterization of the major reactive metabolite in liver microsomes, Chem Res Toxicol 21, 2035-2041. Chen, Y., Monshouwer, M., and Fitch, W. L. (2007) Analytical tools and approaches for metabolite identification in early drug discovery, Pharm Res 24, 248-257. Osorio-Lozada, A., Surapaneni, S., Skiles, G. L., and Subramanian, R. (2008) Biosynthesis of drug metabolites using microbes in hollow fiber cartridge reactors: case study of diclofenac metabolism by Actinoplanes species, Drug Metab Dispos 36, 234-240. Damsten, M. C., van Vugt-Lussenburg, B. M., Zeldenthuis, T., de Vlieger, J. S., Commandeur, J. N., and Vermeulen, N. P. (2008) Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites, Chem Biol Interact 171, 96-107. Damsten, M. C., de Vlieger, J. S., Niessen, W. M., Irth, H., Vermeulen, N. P., and Commandeur, J. N. (2008) Trimethoprim: novel reactive intermediates and bioactivation pathways by cytochrome p450s, Chem Res Toxicol 21, 2181-2187. Boerma, J. S., Vermeulen, N. P., and Commandeur, J. N. (2011) Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites, Chem Res Toxicol 24, 1263-1274. Dragovic, S., Boerma, J. S., van Bergen, L., Vermeulen, N. P., and Commandeur, J. N. (2010) Role of human glutathione S-transferases in the inactivation of reactive metabolites of clozapine, Chem Res Toxicol 23, 1467-1476. Lim, H. K., Stellingweif, S., Sisenwine, S., and Chan, K. W. (1999) Rapid drug metabolite profiling using fast liquid chromatography, automated multiple-stage mass spectrometry and receptor-binding, J Chromatogr A 831, 227-241. Soldner, A., Spahn-Langguth, H., Palm, D., and Mutschler, E. (1998) A radioreceptor assay for the analysis of AT1-receptor antagonists. Correlation with complementary LC data reveals a potential contribution of active metabolites, Journal of pharmaceutical and biomedical analysis 17, 111-124. de Vlieger, J. S., Kolkman, A. J., Ampt, K. A., Commandeur, J. N., Vermeulen, N. P., Kool, J., Wijmenga, S. S., Niessen, W. M., Irth, H., and Honing, M. (2010) Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR, J Chromatogr B Analyt Technol Biomed Life Sci 878, 667-674. Falck, D., de Vlieger, J. S., Niessen, W. M., Kool, J., Honing, M., Giera, M., and Irth, H. (2010) Development of an online p38alpha mitogen-activated protein kinase binding assay and integration of LC-HR-MS, Analytical and bioanalytical chemistry 398, 1771-1780. Reinen, J., Kalma, L. L., Begheijn, S., Heus, F., Commandeur, J. N., and Vermeulen, N. P. (2011) Application of cytochrome P450 BM3 mutants as biocatalysts for the profiling of estrogen receptor binding metabolites of the mycotoxin zearalenone, Xenobiotica; the fate of foreign compounds in biological systems 41, 59-70. Chapter 1 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. General Introduction Miller, K. J., and Herman, A. C. (1996) Affinity chromatography with immunochemical detection applied to the analysis of human methionyl granulocyte colony stimulating factor in serum, Anal Chem 68, 3077-3082. Przyjazny, A., Hentz, N. G., and Bachas, L. G. (1993) Sensitive and selective liquid chromatographic postcolumn reaction detection system for biotin and biocytin using a homogeneous fluorophore-linked assay, J Chromatogr A 654, 79-86. Oosterkamp, A. J., Irth, H., Beth, M., Unger, K. K., Tjaden, U. R., and van de Greef, J. (1994) Bioanalysis of digoxin and its metabolites using direct serum injection combined with liquid chromatography and on-line immunochemical detection, Journal of chromatography. B, Biomedical applications 653, 55-61. Kool, J., van Liempd, S. M., Ramautar, R., Schenk, T., Meerman, J. H., Irth, H., Commandeur, J. N., and Vermeulen, N. P. (2005) Development of a novel cytochrome p450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography, Journal of biomolecular screening 10, 427-436. Ingkaninan, K., Hazekamp, A., de Best, C. M., Irth, H., Tjaden, U. R., van der Heijden, R., van der Greef, J., and Verpoorte, R. (2000) The application of HPLC with on-line coupled UV/MS-biochemical detection for isolation of an acetylcholinesterase inhibitor from narcissus 'Sir Winston Churchill', J Nat Prod 63, 803-806. Schenk, T., Appels, N. M., van Elswijk, D. A., Irth, H., Tjaden, U. R., and van der Greef, J. (2003) A generic assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography for lead finding in natural products, Anal Biochem 316, 118-126. Krabbe, J. G., Lingeman, H., Niessen, W. M., and Irth, H. (2003) Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry, Anal Chem 75, 6853-6860. Van Liempd, S. M., Kool, J., Meerman, J. H., Irth, H., and Vermeulen, N. P. (2007) Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening, Chem Res Toxicol 20, 1825-1832. Giera, M., de Vlieger, J. S., Lingeman, H., Irth, H., and Niessen, W. M. (2010) Structural elucidation of biologically active neomycin N-octyl derivatives in a regioisomeric mixture by means of liquid chromatography/ion trap time-of-flight mass spectrometry, Rapid communications in mass spectrometry : RCM 24, 14391446. Giera, M., Heus, F., Janssen, L., Kool, J., Lingeman, H., and Irth, H. (2009) Microfractionation revisited: a 1536 well high resolution screening assay, Anal Chem 81, 5460-5466. Donova, M. V., and Egorova, O. V. (2012) Microbial steroid transformations: current state and prospects, Appl Microbiol Biotechnol 94, 1423-1447. Hogg, J. A. (1992) Steroids, the steroid community, and Upjohn in perspective: a profile of innovation, Steroids 57, 593-616. Sedlaczek, L. (1988) Biotransformations of steroids, Crit Rev Biotechnol 7, 187236. Chapter 1 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. General Introduction Fernandes, A., Cruz, A., Angelova, B., Pinheiro, H. M., and Cabral, A. (2003) Microbial conversion of steroid compounds: recent developments, Enzyme Microb Technol 32, 688-705. Mahato, S. B., Banerjee, S., and Sahu, N. P. (1984) Metabolism of progesterone and testosterone by a Bacillus sp, Steroids 43, 545-558. Mahato, S. B., and Mukherjee, A. (1984) Microbial transformation of testosterone by Aspergillus fumigatus, J Steroid Biochem 21, 341-342. Ko, D., Heiman, A. S., Chen, M., and Lee, H. J. (2000) New steroidal antiinflammatory antedrugs: methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy3,20-dioxo-1, 4-pregnadiene-16alpha-carboxylate, methyl 21-desoxy-21-chloro11beta-hydroxy-3,20-dioxo-1, 4-pregnadiene-16alpha-carboxylate, and their 9alpha-fluoro derivatives*, Steroids 65, 210-218. Tuba, Z., Bardin, C. W., Dancsi, A., Francsics-Czinege, E., Molnar, C., Csorgei, J., Falkay, G., Koide, S. S., Kumar, N., Sundaram, K., Dukat-Abrok, V., and Balogh, G. (2000) Synthesis and biological activity of a new progestogen, 16-methylene17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate, Steroids 65, 266-274. Newhouse, T., and Baran, P. S. (2011) If C-H bonds could talk: selective C-H bond oxidation, Angew Chem Int Ed Engl 50, 3362-3374. Dutton, D. R., McMillen, S. K., Sonderfan, A. J., Thomas, P. E., and Parkinson, A. (1987) Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions, Archives of biochemistry and biophysics 255, 316328. Sonderfan, A. J., Arlotto, M. P., and Parkinson, A. (1989) Identification of the cytochrome P-450 isozymes responsible for testosterone oxidation in rat lung, kidney, and testis: evidence that cytochrome P-450a (P450IIA1) is the physiologically important testosterone 7 alpha-hydroxylase in rat testis, Endocrinology 125, 857-866. Agematu, H., Matsumoto, N., Fujii, Y., Kabumoto, H., Doi, S., Machida, K., Ishikawa, J., and Arisawa, A. (2006) Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system, Bioscience, biotechnology, and biochemistry 70, 307-311. Bernhardt, R. (2006) Cytochromes P450 as versatile biocatalysts, J Biotechnol 124, 128-145. Hannemann, F., Bichet, A., Ewen, K. M., and Bernhardt, R. (2007) Cytochrome P450 systems--biological variations of electron transport chains, Biochim Biophys Acta 1770, 330-344. Novikova, L. A., Faletrov, Y. V., Kovaleva, I. E., Mauersberger, S., Luzikov, V. N., and Shkumatov, V. M. (2009) From structure and functions of steroidogenic enzymes to new technologies of gene engineering, Biochemistry. Biokhimiia 74, 1482-1504. Kristan, K., and Rizner, T. L. (2012) Steroid-transforming enzymes in fungi, The Journal of steroid biochemistry and molecular biology 129, 79-91. Guengerich, F. P. (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity, Chem Res Toxicol 14, 611-650. Chapter 1 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. General Introduction O'Reilly, E., Kohler, V., Flitsch, S. L., and Turner, N. J. (2010) Cytochromes P450 as useful biocatalysts: addressing the limitations, Chem Commun (Camb) 47, 2490-2501. Drnelson.utmem.edu. Pirmohamed, M., and Park, B. K. (2003) Cytochrome P450 enzyme polymorphisms and adverse drug reactions, Toxicology 192, 23-32. Ingelman-Sundberg, M. (2004) Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future, Trends in pharmacological sciences 25, 193-200. Kirchheiner, J., and Brockmoller, J. (2005) Clinical consequences of cytochrome P450 2C9 polymorphisms, Clin Pharmacol Ther 77, 1-16. Urlacher, V. B., and Girhard, M. (2011) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application, Trends Biotechnol 30, 26-36. Munro, A. W., Leys, D. G., McLean, K. J., Marshall, K. R., Ost, T. W., Daff, S., Miles, C. S., Chapman, S. K., Lysek, D. A., Moser, C. C., Page, C. C., and Dutton, P. L. (2002) P450 BM3: the very model of a modern flavocytochrome, Trends Biochem Sci 27, 250-257. Isin, E. M., and Guengerich, F. P. (2007) Complex reactions catalyzed by cytochrome P450 enzymes, Biochim Biophys Acta 1770, 314-329. Anzenbacher, P., and Anzenbacherova, E. (2001) Cytochromes P450 and metabolism of xenobiotics, Cell Mol Life Sci 58, 737-747. Whitehouse, C. J., Bell, S. G., and Wong, L. L. (2012) P450(BM3) (CYP102A1): connecting the dots, Chemical Society reviews 41, 1218-1260. Rittle, J., and Green, M. T. (2010) Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics, Science 330, 933-937. Loida, P. J., and Sligar, S. G. (1993) Molecular recognition in cytochrome P-450: mechanism for the control of uncoupling reactions, Biochemistry 32, 1153011538. Lussenburg, B. M., Babel, L. C., Vermeulen, N. P., and Commandeur, J. N. (2005) Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants, Anal Biochem 341, 148-155. Jung, S. T., Lauchli, R., and Arnold, F. H. (2011) Cytochrome P450: taming a wild type enzyme, Curr Opin Biotechnol 22, 809-817. Warman, A. J., Roitel, O., Neeli, R., Girvan, H. M., Seward, H. E., Murray, S. A., McLean, K. J., Joyce, M. G., Toogood, H., Holt, R. A., Leys, D., Scrutton, N. S., and Munro, A. W. (2005) Flavocytochrome P450 BM3: an update on structure and mechanism of a biotechnologically important enzyme, Biochem Soc Trans 33, 747-753. Narhi, L. O., and Fulco, A. J. (1986) Characterization of a catalytically selfsufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium, J Biol Chem 261, 7160-7169. Carmichael, A. B., and Wong, L. L. (2001) Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons, Eur J Biochem 268, 3117-3125. Chapter 1 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. General Introduction Dietrich, M., Do, T. A., Schmid, R. D., Pleiss, J., and Urlacher, V. B. (2009) Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions, J Biotechnol 139, 115-117. Seifert, A., Vomund, S., Grohmann, K., Kriening, S., Urlacher, V. B., Laschat, S., and Pleiss, J. (2009) Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity, Chembiochem 10, 853-861. Munzer, D. F., Meinhold, P., Peters, M. W., Feichtenhofer, S., Griengl, H., Arnold, F. H., Glieder, A., and de Raadt, A. (2005) Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM3, Chem Commun (Camb), 2597-2599. Kim, D. H., Ahn, T., Jung, H. C., Pan, J. G., and Yun, C. H. (2009) Generation of the human metabolite piceatannol from the anticancer-preventive agent resveratrol by bacterial cytochrome P450 BM3, Drug Metab Dispos 37, 932-936. Kim, D. H., Kim, K. H., Liu, K. H., Jung, H. C., Pan, J. G., and Yun, C. H. (2008) Generation of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3, Drug Metab Dispos 36, 2166-2170. Kim, K. H., Kang, J. Y., Kim, D. H., Park, S. H., Kim, D., Park, K. D., Lee, Y. J., Jung, H. C., Pan, J. G., Ahn, T., and Yun, C. H. (2011) Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants, Drug Metab Dispos 39, 140-150. Landwehr, M., Hochrein, L., Otey, C. R., Kasrayan, A., Backvall, J. E., and Arnold, F. H. (2006) Enantioselective alpha-hydroxylation of 2-arylacetic acid derivatives and buspirone catalyzed by engineered cytochrome P450 BM-3, J Am Chem Soc 128, 6058-6059. Sawayama, A. M., Chen, M. M., Kulanthaivel, P., Kuo, M. S., Hemmerle, H., and Arnold, F. H. (2009) A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds, Chemistry 15, 11723-11729. van Vugt-Lussenburg, B. M., Damsten, M. C., Maasdijk, D. M., Vermeulen, N. P., and Commandeur, J. N. (2006) Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3, Biochem Biophys Res Commun 346, 810-818. van Vugt-Lussenburg, B. M., Stjernschantz, E., Lastdrager, J., Oostenbrink, C., Vermeulen, N. P., and Commandeur, J. N. (2007) Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis, J Med Chem 50, 455-461. Rea, V., Dragovic, S., Boerma, J. S., de Kanter, F. J., Vermeulen, N. P., and Commandeur, J. N. (2011) Role of residue 87 in the activity and regioselectivity of clozapine metabolism by drug-metabolizing CYP102A1 M11H: application for structural characterization of clozapine GSH conjugates, Drug Metab Dispos 39, 2411-2420. Yun, C. H., Kim, K. H., Kim, D. H., Jung, H. C., and Pan, J. G. (2007) The bacterial P450 BM3: a prototype for a biocatalyst with human P450 activities, Trends Biotechnol 25, 289-298. Gillam, E. M. (2008) Engineering cytochrome p450 enzymes, Chem Res Toxicol 21, 220-231. Chapter 1 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. General Introduction Papouchado, B. G., Myles, J., Lloyd, R. V., Stoler, M., Oliveira, A. M., DownsKelly, E., Morey, A., Bilous, M., Nagle, R., Prescott, N., Wang, L., Dragovich, L., McElhinny, A., Garcia, C. F., Ranger-Moore, J., Free, H., Powell, W., Loftus, M., Pettay, J., Gaire, F., Roberts, C., Dietel, M., Roche, P., Grogan, T., and Tubbs, R. (2010) Silver in situ hybridization (SISH) for determination of HER2 gene status in breast carcinoma: comparison with FISH and assessment of interobserver reproducibility, The American journal of surgical pathology 34, 767-776. Feenstra, K. A., Hofstetter, K., Bosch, R., Schmid, A., Commandeur, J. N., and Vermeulen, N. P. (2006) Enantioselective substrate binding in a monooxygenase protein model by molecular dynamics and docking, Biophysical journal 91, 32063216. Rea, V., Kolkman, A. J., Vottero, E. R., Stronks, E. J., Ampt, K. A., Honing, M., Vermeulen, N. P., Wijmenga, S. S., and Commandeur, J. N. (2011) Active site substitution A82W improves regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalised by spin relaxation NMR studies, Biochemistry. Huang, W. C., Westlake, A. C., Marechal, J. D., Joyce, M. G., Moody, P. C., and Roberts, G. C. (2007) Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency, J Mol Biol 373, 633-651. Dietrich, J. A., Yoshikuni, Y., Fisher, K. J., Woolard, F. X., Ockey, D., McPhee, D. J., Renninger, N. S., Chang, M. C., Baker, D., and Keasling, J. D. (2009) A novel semi-biosynthetic route for artemisinin production using engineered substratepromiscuous P450(BM3), ACS chemical biology 4, 261-267. Seifert, A., Antonovici, M., Hauer, B., and Pleiss, J. (2011) An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1, Chembiochem 12, 1346-1351. Koenig, R., Lesemann, D. E., Loss, S., Engelmann, J., Commandeur, U., Deml, G., Schiemann, J., Aust, H., and Burgermeister, W. (2006) Zygocactus virus X-based expression vectors and formation of rod-shaped virus-like particles in plants by the expressed coat proteins of Beet necrotic yellow vein virus and Soil-borne cereal mosaic virus, The Journal of general virology 87, 439-443. Julsing, M. K., Cornelissen, S., Buhler, B., and Schmid, A. (2008) Heme-iron oxygenases: powerful industrial biocatalysts?, Curr Opin Chem Biol 12, 177-186. Parikh, A., Gillam, E. M., and Guengerich, F. P. (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450, Nat Biotechnol 15, 784788. Rushmore, T. H., Reider, P. J., Slaughter, D., Assang, C., and Shou, M. (2000) Bioreactor systems in drug metabolism: synthesis of cytochrome P450-generated metabolites, Metab Eng 2, 115-125. Dubey, K. K., and Behera, B. K. (2011) Statistical optimization of process variables for the production of an anticancer drug (colchicine derivatives) through fermentation: at scale-up level, New biotechnology 28, 79-85. Lewis, J. C., Bastian, S., Bennett, C. S., Fu, Y., Mitsuda, Y., Chen, M. M., Greenberg, W. A., Wong, C. H., and Arnold, F. H. (2009) Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases, Proc Natl Acad Sci U S A 106, 16550-16555. Chapter 1 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. General Introduction Rentmeister, A., Arnold, F. H., and Fasan, R. (2009) Chemo-enzymatic fluorination of unactivated organic compounds, Nat Chem Biol 5, 26-28. Ferrero, V. E., Di Nardo, G., Catucci, G., Sadeghi, S. J., and Gilardi, G. (2012) Fluorescence detection of ligand binding to labeled cytochrome P450 BM3, Dalton Trans 41, 2018-2025. Vottero, E., Rea, V., Lastdrager, J., Honing, M., Vermeulen, N. P., and Commandeur, J. N. (2011) Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11, J Biol Inorg Chem 16, 899-912. Okubo, Y., Bessho, K., Fujimura, K., Kusumoto, K., Ogawa, Y., and Iizuka, T. (2002) Expression of bone morphogenetic protein in the course of osteoinduction by recombinant human bone morphogenetic protein-2, Clinical oral implants research 13, 80-85. Ogawa, M., Kumagai, K., Gondo, N., Matsumoto, N., Suyama, K., and Saku, K. (2002) Novel electrophysiologic parameter of dispersion of atrial repolarization: comparison of different atrial pacing methods, J Cardiovasc Electrophysiol 13, 110-117. Fedorova, A., and Ogawa, M. Y. (2002) Site-specific modification of de novo designed coiled-coil polypeptides with inorganic redox complexes, Bioconjug Chem 13, 150-154. Hoehn, R., Lamparter, J., Pfeiffer, N., and Vossmerbaeumer, U. (2011) Seizures following subconjunctival 5-FU therapy, Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie 249, 145-146. Graham-Lorence, S., Truan, G., Peterson, J. A., Falck, J. R., Wei, S., Helvig, C., and Capdevila, J. H. (1997) An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase, J Biol Chem 272, 1127-1135. Oliver, C. F., Modi, S., Sutcliffe, M. J., Primrose, W. U., Lian, L. Y., and Roberts, G. C. (1997) A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation, Biochemistry 36, 1567-1572. Nulens, E., Gould, I., MacKenzie, F., Deplano, A., Cookson, B., Alp, E., Bouza, E., and Voss, A. (2005) Staphylococcus aureus carriage among participants at the 13th European Congress of Clinical Microbiology and Infectious Diseases, European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 24, 145-148. Schotten, U., Schumacher, C., Conrads, V., Braun, V., Schondube, F., Voss, M., and Hanrath, P. (1999) Calcium-sensitivity of the SR calcium release channel in failing and nonfailing human myocardium, Basic research in cardiology 94, 145151. de Smet, M. D., Buffam, F. V., Fairey, R. N., and Voss, N. J. (1990) Prevention of radiation-induced stenosis of the nasolacrimal duct, Canadian journal of ophthalmology. Journal canadien d'ophtalmologie 25, 145-147. Voss, K., Barz, H., and Wagner, H. (1977) [Image processing in pathology. III. Structure and use of an automatic morphometry system for recording Chapter 1 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. General Introduction morphometric features of nuclei (author's transl)], Experimentelle Pathologie 13, 145-161. Voss, R., Rickart, A., Ruile, K., Schoen, H. R., and Kunz, W. (1970) [Hyperbaric organ preservation], Verhandlungen der Deutschen Gesellschaft fur Pathologie 54, 145-150. Vossschulte, K. (1951) [The intervertebral disks as affected by the movements of the spine], Langenbecks Archiv fur klinische Chirurgie ... vereinigt mit Deutsche Zeitschrift fur Chirurgie 267, 145. Ruperti, B., Bonghi, C., Rasori, A., Ramina, A., and Tonutti, P. (2001) Characterization and expression of two members of the peach 1aminocyclopropane-1-carboxylate oxidase gene family, Physiol Plant 111, 336344. Bidoli, V., Casolino, M., De Pascale, M. P., Furano, G., Minori, M., Morselli, A., Narici, L., Picozza, P., Reali, E., Sparvoli, R., Fuglesang, C., Sannita, W., Carlson, P., Castellini, G., Galper, A., Korotkov, M., Popov, A., Navilov, N., Avdeev, S., Benghin, V., Salnitskii, V., Shevchenko, O., Boezio, M., Bonvicini, W., Vacchi, A., Zampa, G., Zampa, N., Mazzenga, G., Ricci, M., Spillantini, P., and Vittori, R. (2002) The Sileye-3/Alteino experiment for the study of light flashes, radiation environment and astronaut brain activity on board the International Space Station, Journal of radiation research 43 Suppl, S47-52. Furalev, V. A., Kravchenko, I. V., Vasil'eva, L. A., and Pylev, L. N. (2002) Cytotoxic effects of macrophages and asbestos on transformed rat mesothelial cells, Bulletin of experimental biology and medicine 133, 71-73. Walldal, E., Anund, I., and Furaker, C. (2002) Quality of care and development of a critical pathway, Journal of nursing management 10, 115-122. Li, Q. S., Schwaneberg, U., Fischer, P., and Schmid, R. D. (2000) Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst, Chemistry 6, 1531-1536. Li, Q. S., Ogawa, J., Schmid, R. D., and Shimizu, S. (2001) Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatic hydrocarbons, Appl Environ Microbiol 67, 5735-5739. Volz, T. J., Rock, D. A., and Jones, J. P. (2002) Evidence for two different active oxygen species in cytochrome P450 BM3 mediated sulfoxidation and Ndealkylation reactions, J Am Chem Soc 124, 9724-9725. Chen, C. K., Shokhireva, T., Berry, R. E., Zhang, H., and Walker, F. A. (2008) The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity, J Biol Inorg Chem 13, 813-824. Eiben, S., Kaysser, L., Maurer, S., Kuhnel, K., Urlacher, V. B., and Schmid, R. D. (2006) Preparative use of isolated CYP102 monooxygenases -- a critical appraisal, J Biotechnol 124, 662-669. Rock, D. A., Perkins, B. N., Wahlstrom, J., and Jones, J. P. (2003) A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation, Archives of biochemistry and biophysics 416, 9-16. Whitehouse, C. J., Yang, W., Yorke, J. A., Tufton, H. G., Ogilvie, L. C., Bell, S. G., Zhou, W., Bartlam, M., Rao, Z., and Wong, L. L. (2011) Structure, electronic Chapter 1 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. General Introduction properties and catalytic behaviour of an activity-enhancing CYP102A1 (P450(BM3)) variant, Dalton Trans 40, 10383-10396. Haines, D. C., Tomchick, D. R., Machius, M., and Peterson, J. A. (2001) Pivotal role of water in the mechanism of P450BM-3, Biochemistry 40, 13456-13465. Rock, D. A., Boitano, A. E., Wahlstrom, J. L., and Jones, J. P. (2002) Use of kinetic isotope effects to delineate the role of phenylalanine 87 in P450(BM-3), Bioorganic chemistry 30, 107-118. Feenstra, K. A., Starikov, E. B., Urlacher, V. B., Commandeur, J. N., and Vermeulen, N. P. (2007) Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants, Protein science : a publication of the Protein Society 16, 420-431. Wong, T. S., Arnold, F. H., and Schwaneberg, U. (2004) Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents, Biotechnol Bioeng 85, 351-358. Roccatano, D., Wong, T. S., Schwaneberg, U., and Zacharias, M. (2006) Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: a molecular dynamics simulation study, Biopolymers 83, 467476. Urlacher, V. B., Makhsumkhanov, A., and Schmid, R. D. (2006) Biotransformation of beta-ionone by engineered cytochrome P450 BM-3, Appl Microbiol Biotechnol 70, 53-59. Sowden, R. J., Yasmin, S., Rees, N. H., Bell, S. G., and Wong, L. L. (2005) Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3, Org Biomol Chem 3, 57-64. Whitehouse, C. J., Bell, S. G., Tufton, H. G., Kenny, R. J., Ogilvie, L. C., and Wong, L. L. (2008) Evolved CYP102A1 (P450BM3) variants oxidise a range of non-natural substrates and offer new selectivity options, Chem Commun (Camb), 966-968. Wong, T. S., Wu, N., Roccatano, D., Zacharias, M., and Schwaneberg, U. (2005) Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds, Journal of biomolecular screening 10, 246-252. Chen, C. K., Berry, R. E., Shokhireva, T., Murataliev, M. B., Zhang, H., and Walker, F. A. (2010) Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene omega-hydroxylase CYP4C7, J Biol Inorg Chem 15, 159-174. Reinen, J., Ferman, S., Vottero, E., Vermeulen, N. P., and Commandeur, J. N. (2011) Application of a fluorescence-based continuous-flow bioassay to screen for diversity of cytochrome P450 BM3 mutant libraries, Journal of biomolecular screening 16, 239-250. Noble, M. A., Miles, C. S., Chapman, S. K., Lysek, D. A., MacKay, A. C., Reid, G. A., Hanzlik, R. P., and Munro, A. W. (1999) Roles of key active-site residues in flavocytochrome P450 BM3, Biochem J 339 ( Pt 2), 371-379. Raner, G. M., Hatchell, J. A., Dixon, M. U., Joy, T. L., Haddy, A. E., and Johnston, E. R. (2002) Regioselective peroxo-dependent heme alkylation in P450(BM3)F87G by aromatic aldehydes: effects of alkylation on cataysis, Biochemistry 41, 9601-9610. Chapter 1 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. General Introduction Brenner, S., Hay, S., Girvan, H. M., Munro, A. W., and Scrutton, N. S. (2007) Conformational dynamics of the cytochrome P450 BM3/N-palmitoylglycine complex: the proposed "proximal-distal" transition probed by temperature-jump spectroscopy, J Phys Chem B 111, 7879-7886. Raner, G. M., Thompson, J. I., Haddy, A., Tangham, V., Bynum, N., Ramachandra Reddy, G., Ballou, D. P., and Dawson, J. H. (2006) Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors, J Inorg Biochem 100, 2045-2053. Noble, M. A., Quaroni, L., Chumanov, G. D., Turner, K. L., Chapman, S. K., Hanzlik, R. P., and Munro, A. W. (1998) Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3, Biochemistry 37, 15799-15807. Branco, R. J., Seifert, A., Budde, M., Urlacher, V. B., Ramos, M. J., and Pleiss, J. (2008) Anchoring effects in a wide binding pocket: the molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium, Proteins 73, 597-607. Li, Q. S., Ogawa, J., Schmid, R. D., and Shimizu, S. (2001) Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation, FEBS Lett 508, 249-252. Waltham, T. N., Girvan, H. M., Butler, C. F., Rigby, S. R., Dunford, A. J., Holt, R. A., and Munro, A. W. (2011) Analysis of the oxidation of short chain alkynes by flavocytochrome P450 BM3, Metallomics 3, 369-378. Meinhold, P., Peters, M. W., Chen, M. M., Takahashi, K., and Arnold, F. H. (2005) Direct conversion of ethane to ethanol by engineered cytochrome P450 BM3, Chembiochem 6, 1765-1768. Kubo, T., Peters, M. W., Meinhold, P., and Arnold, F. H. (2006) Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3, Chemistry 12, 1216-1220. Kitazume, T., Haines, D. C., Estabrook, R. W., Chen, B., and Peterson, J. A. (2007) Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM3, Biochemistry 46, 11892-11901. Daiber, A., Herold, S., Schoneich, C., Namgaladze, D., Peterson, J. A., and Ullrich, V. (2000) Nitration and inactivation of cytochrome P450BM-3 by peroxynitrite. Stopped-flow measurements prove ferryl intermediates, Eur J Biochem 267, 6729-6739. Cowart, L. A., Falck, J. R., and Capdevila, J. H. (2001) Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3, Archives of biochemistry and biophysics 387, 117-124. Li, Q. S., Ogawa, J., Schmid, R. D., and Shimizu, S. (2005) Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide, Bioscience, biotechnology, and biochemistry 69, 293-300. Lewis, J. C., Mantovani, S. M., Fu, Y., Snow, C. D., Komor, R. S., Wong, C. H., and Arnold, F. H. (2010) Combinatorial alanine substitution enables rapid optimization of cytochrome P450BM3 for selective hydroxylation of large substrates, Chembiochem 11, 2502-2505. Chapter 1 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. General Introduction Weber, E., Seifert, A., Antonovici, M., Geinitz, C., Pleiss, J., and Urlacher, V. B. Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes, Chem Commun (Camb) 47, 944-946. Sulistyaningdyah, W. T., Ogawa, J., Li, Q. S., Maeda, C., Yano, Y., Schmid, R. D., and Shimizu, S. (2005) Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds, Appl Microbiol Biotechnol 67, 556-562. Misawa, N., Nodate, M., Otomatsu, T., Shimizu, K., Kaido, C., Kikuta, M., Ideno, A., Ikenaga, H., Ogawa, J., Shimizu, S., and Shindo, K. (2011) Bioconversion of substituted naphthalenes and beta-eudesmol with the cytochrome P450 BM3 variant F87V, Appl Microbiol Biotechnol 90, 147-157. Nazor, J., and Schwaneberg, U. (2006) Laboratory evolution of P450 BM-3 for mediated electron transfer, Chembiochem 7, 638-644. Fasan, R., Chen, M. M., Crook, N. C., and Arnold, F. H. (2007) Engineered alkanehydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties, Angew Chem Int Ed Engl 46, 8414-8418. Peters, M. W., Meinhold, P., Glieder, A., and Arnold, F. H. (2003) Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3, J Am Chem Soc 125, 13442-13450. Murataliev, M. B., Trinh, L. N., Moser, L. V., Bates, R. B., Feyereisen, R., and Walker, F. A. (2004) Chimeragenesis of the fatty acid binding site of cytochrome P450BM3. Replacement of residues 73-84 with the homologous residues from the insect cytochrome P450 CYP4C7, Biochemistry 43, 1771-1780. Huang, W. C., Cullis, P. M., Raven, E. L., and Roberts, G. C. (2011) Control of the stereo-selectivity of styrene epoxidation by cytochrome P450 BM3 using structure-based mutagenesis, Metallomics 3, 410-416. Rea, V., Kolkman, A. J., Vottero, E., Stronks, E. J., Ampt, K. A., Honing, M., Vermeulen, N. P., Wijmenga, S. S., and Commandeur, J. N. (2012) Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies, Biochemistry 51, 750-760. Venkataraman, H., Beer, S. B., Bergen, L. A., Essen, N., Geerke, D. P., Vermeulen, N. P., and Commandeur, J. N. (2012) A single active site mutation inverts stereoselectivity of 16-hydroxylation of testosterone catalyzed by engineered cytochrome P450 BM3, Chembiochem 13, 520-523. Novak, R. F., and Vatsis, K. P. (1982) 1H Fourier transform nuclear magnetic resonance relaxation rate studies on the interaction of acetanilide with purified isozymes of rabbit liver microsomal cytochrome P-450 and with cytochrome b5, Mol Pharmacol 21, 701-709. Solomon, I., and Bloembergen, N. (1956) Nuclear magnetic interactions in the HF molecule, J Chem Phys 25, 261-266. Regal, K. A., and Nelson, S. D. (2000) Orientation of caffeine within the active site of human cytochrome P450 1A2 based on NMR longitudinal (T1) relaxation measurements, Archives of biochemistry and biophysics 384, 47-58. van de Straat, R., de Vries, J., de Boer, H. J., Vromans, R. M., and Vermeulen, N. P. (1987) Relationship between paracetamol binding to and its oxidation by two Chapter 1 165. 166. 167. 168. 169. General Introduction cytochromes P-450 isozymes--a proton nuclear magnetic resonance and spectrophotometric study, Xenobiotica; the fate of foreign compounds in biological systems 17, 1-9. Poli-Scaife, S., Attias, R., Dansette, P. M., and Mansuy, D. (1997) The substrate binding site of human liver cytochrome P450 2C9: an NMR study, Biochemistry 36, 12672-12682. Modi, S., Paine, M. J., Sutcliffe, M. J., Lian, L. Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. (1996) A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding, Biochemistry 35, 4540-4550. Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L. Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. (1997) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase, Biochemistry 36, 4461-4470. Smith, G., Modi, S., Pillai, I., Lian, L. Y., Sutcliffe, M. J., Pritchard, M. P., Friedberg, T., Roberts, G. C., and Wolf, C. R. (1998) Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity, Biochem J 331 ( Pt 3), 783-792. Keizers, P. H., de Graaf, C., de Kanter, F. J., Oostenbrink, C., Feenstra, K. A., Commandeur, J. N., and Vermeulen, N. P. (2005) Metabolic regio- and stereoselectivity of cytochrome P450 2D6 towards 3,4-methylenedioxy-Nalkylamphetamines: in silico predictions and experimental validation, J Med Chem 48, 6117-6127