Human P450-like oxidation of diverse proton pump inhibitor drugs by 'gatekeeper' mutants of flavocytochrome P450 BM3.

Biochem. J. (2014) 460, 247–259 (Printed in Great Britain)

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doi:10.1042/BJ20140030

*Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. †Cypex Ltd, 6 Tom McDonald Avenue, Dundee DD2 1NH, U.K. ‡Agilent Technologies UK Ltd, Lakeside, Cheadle Royal Business Park, Stockport, Cheshire SK8 3GR, U.K.

Production of drug metabolites is one area where enzymatic conversion has significant advantages over synthetic chemistry. These high value products are complex to synthesize, but are increasingly important in drug safety testing. The vast majority of drugs are metabolized by cytochromes P450 (P450s), with oxidative transformations usually being highly regio- and stereoselective. The PPIs (proton pump inhibitors) are drugs that are extensively metabolized by human P450s, producing diverse metabolites dependent on the specific substrate. In the present paper we show that single mutations (A82F and F87V) in the biotechnologically important Bacillus megaterium P450 BM3 enzyme cause major alterations in its substrate selectivity such that a set of PPI molecules become good substrates in these point mutants and in the F87V/A82F double mutant. The substrate

specificity switch is analysed by drug binding, enzyme kinetics and organic product analysis to confirm new activities, and Xray crystallography provides a structural basis for the binding of esomeprazole to the F87V/A82F enzyme. These studies confirm that such ‘gatekeeper’ mutations in P450 BM3 produce major perturbations to its conformation and substrate selectivity, enabling novel P450 BM3 reactions typical of those performed by human P450s. Efficient transformation of several PPI drugs to human-like products by BM3 variants provides new routes to production of these metabolites.

INTRODUCTION

both catabolic (e.g. initiation of the breakdown of vitamin D3 by human CYP24A1) and anabolic (e.g. cholesterol synthesis by CYP51 enzymes) processes (e.g. [5,7,8]). Human P450s are responsible for most of the primary (phase I) xenobiotic metabolism in humans, producing oxidized metabolites of pharmaceuticals that are then more readily excreted, or else further modified and targeted for excretion by phase II enzymes such as glutathione transferases. However, P450s are also responsible for the generation of activated drugs from their prodrug forms (e.g. the oxidation of the anti-cancer drug ellipticine by human CYP3A4 to generate DNA-modifying products) [9]. In recent years, the synthetic potential of the P450s has been recognized, and various P450s have been targets for biotechnological exploitation and subject to the diversification of their substrate recognition and the regio-/stereo-selectivity of their substrate oxidation by protein engineering methods such as chimaeragenesis and directed evolution (e.g. [10,11]). The most promising P450 for biotechnological applications is the Bacillus megaterium P450/CPR (P450 reductase) fusion enzyme P450 BM3 (BM3; also known as CYP102A1), due to its high expression levels, soluble nature (compared with membranebound eukaryotic P450s) and convenient single component organization (P450 and CPR domains on the same polypeptide chain). Its efficient electron transfer system (for electron transfer both within the CPR domain and between the CPR and the P450 domain) results in BM3 having the highest reported catalytic rate of substrate oxidation among the P450s (e.g. 17000 min − 1

Replacing traditional synthetic routes to complex organic molecules using enzyme-catalysed reactions has many advantages. These include lower energy requirements, fewer synthetic steps and the ability to activate centres that cannot be readily activated by traditional chemical synthesis methods. A promising area for this research is in the production of fine chemicals and drug metabolites (e.g. [1–3]). Human drug metabolites are often complex molecules that are expensive to prepare chemically, and thus attractive candidates for the application of enzymatic synthesis, given their high value as standards and as reagents in drug testing for the biotechnology and pharmaceutical industries [4]. Cytochromes P450 (P450s or CYPs) are a superfamily of haem-containing monooxygenase enzymes that are of particular interest for synthetic applications, due to their ability to activate dioxygen and to insert one of its oxygen atoms into an unactivated CH bond, with the second oxygen atom being reduced to water [5]. P450s catalyse numerous physiologically important reactions (e.g. oxidation of arachidonic acid to epoxyeicosatrienoic acids that have vasodilatory and anti-inflammatory effects in mammals) [6], and are also increasingly exploited for natural and unnatural oxidative reactions with potential uses in, for example medicine, bioremediation and pharmacology. These include hydroxylation of fatty acids/steroids, epoxidation of styrene, dealkylation of various drugs, and applications in

Key words: crystal structure, cytochrome P450, drug metabolism, protein engineering, proton pump inhibitor.

Abbreviations: ACN, acetonitrile; BM3, P450 BM3; CPR, P450 reductase; CYP, cytochrome P450; DM, double mutant; ESO, esomeprazole; HMBC, heteronuclear multiple bond correlation; HS, high spin; IS, internal standard; KPi, potassium phosphate; LAN, lansoprazole; LS, low spin; NPG, N -palmitoylglycine; OMP, omeprazole; PAN, pantoprazole; PPI, proton pump inhibitor; P450, CYP P450; RAB, rabeprazole; TB, terrific broth; TMS, tetramethylsilane; WT, wild-type. 1 To whom correspondence should be addressed (email [email protected]). The structural co-ordinates reported for ESO bound to the DM BM3 haem domain have been deposited in the PDB under code 4O4P. c The Authors Journal compilation c 2014 Biochemical Society

Biochemical Journal

Christopher F. BUTLER*, Caroline PEET†, Kirsty J. MCLEAN*, Michael T. BAYNHAM‡, Richard T. BLANKLEY‡, Karl FISHER*, Stephen E. J. RIGBY*, David LEYS*, Michael W. VOICE† and Andrew W. MUNRO*1

www.biochemj.org

Human P450-like oxidation of diverse proton pump inhibitor drugs by ‘gatekeeper’ mutants of flavocytochrome P450 BM3

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C. F. Butler and others

with arachidonic acid) [12]. Engineering of BM3 has generated variants capable of many different reactions, including studies to create an olefin cyclopropanation catalyst [13], and to enable production of oxidized steroids and the oxidation of short chain fatty acids [14,15]. Approaches to altering BM3 substrate selectivity have often relied on the use of random mutagenesis, mostly by error-prone PCR (e.g. [16]). Although this methodology has identified several mutants with novel catalytic properties, it can be a rather random approach to new catalyst generation. BM3 mutants generated by directed evolution approaches typically have numerous mutations dispersed across the P450 domain [17]. However, through analysis of data from several preceding studies on BM3 enzymes evolved for diverse activities, it has become clear that a small number of P450 (haem) domain amino acids are mutated most frequently in variants with novel activities, including those at Ala82 and Phe87 [18]. Previously, we showed that the A82F mutation causes structural destabilization of the BM3 haem domain, resulting in a lower melting temperature, but also in an altered substrate specificity profile. X-ray crystallography showed that the substrate-free A82F haem domain also occupied a conformation different to that of the substrate-free WT (wild-type) P450 [19]. The human gastric PPI (proton pump inhibitor) drug OMP (omeprazole) became a good substrate for the A82F BM3 enzyme, and our structural data revealed the productive mode of binding for OMP that enables its oxidation by the BM3 A82F mutant at the same position as that catalysed by its major human metabolizing P450 CYP2C19 [19]. Binding of OMP was further improved by the additional F87V mutation, where removal of the aromatic bulk of Phe87 frees space immediately in the vicinity of the BM3 haem iron in the P450 active site [19]. In the present study, we demonstrate that mutant BM3 P450s containing the Ala82 and Phe87 mutations also facilitate the binding and oxidation of a range of other PPI drugs [ESO (esomeprazole), LAN (lansoprazole), PAN (pantoprazole) and RAB (rabeprazole)] that are not effective substrates for the WT BM3. The products of oxidation of these drugs are diverse from those of OMP but, as found for OMP, in most cases mimic those products produced by the major human P450s that metabolize these drugs. Our data provide further evidence that mutations at a small number of residues which can impact significantly on the structural organization of BM3 (‘gatekeeper’ mutants) are sufficient to induce major alterations in the substrate selectivity profile of this biotechnologically important P450 enzyme. Thus we demonstrate that such BM3 mutants can produce human-like metabolites of a number of PPI pharmaceuticals, with potential uses for these metabolites as standards and as reagents for pharmaceutical testing and FDA (Food and Drug Administration) compliance. MATERIALS AND METHODS Mutagenesis and expression of WT and mutant BM3 enzymes

The CYP102A1 gene encoding intact WT flavocytochrome BM3 in the plasmid vector pET15b (Novagen) was used for mutagenesis to create A82F, F87V and F87V/A82F [DM (double mutant)] mutants as described previously [19]. Intact BM3 enzymes were expressed as N-terminal His6 -tagged enzymes either from pET15b (F87V and DM) constructs directly or after cloning the WT and A82F genes into pET14b using NdeI/BamHI sites. The WT and mutant haem domain genes were generated using the relevant pET14b/15b constructs as described previously [19]. The haem domain genes (amino acids 1–473 of the 1048 amino acid flavocytochrome) were transferred as NdeI/BamHI fragments into pET20b to allow haem domain production in the absence of an N-terminal His tag and to enable improved c The Authors Journal compilation c 2014 Biochemical Society

protein crystallization. All genes were fully sequenced to confirm relevant mutations and to ensure no exogenous mutations were incorporated. The WT and A82F intact BM3, and WT and all mutant BM3 haem domains, were expressed in BL21 Gold (DE3) Escherichia coli cells (Agilent) in TB (terrific broth) medium with cells grown at 37 ◦ C, and with agitation at 200 rev./min in an orbital incubator. F87V and DM intact BM3 proteins were grown using autoinduction TB medium (Melford) from 4 litre transformant cultures and with cell growth for 24–36 h. Purification of WT and mutant intact P450 BM3 and haem domains

Intact WT and mutant P450 BM3 enzymes and haem domains were purified essentially as described previously [19]. Cells were collected by centrifugation at 4 ◦ C (6000 g for 10 min) and resuspended in ice-cold buffer B [50 mM KPi (potassium phosphate), 250 mM NaCl and 10 % (v/v) glycerol, (pH 7.0)]. Protease inhibitors (EDTA-free CompleteTM tablets; Roche) were maintained in all buffers used during purification. Cells were lysed by sonication on ice using a Bandelin Sonopuls sonicator (at 40 % power, with 50 pulses for 5 s each and 25 s between pulses), and the supernatant containing soluble intact BM3 or haem domain protein was collected after high speed centrifugation (20 000 g for 40 min at 4 ◦ C). The supernatant was collected again after a 30 % ammonium sulfate cut on ice. P450 proteins were purified by Ni-IDA (Ni2 + -iminodiacetic acid) chromatography (Qiagen), with bound proteins washed extensively at 4 ◦ C in buffer B plus 5 mM imidazole, then eluted with 200 mM imidazole in buffer B. Proteins thus purified were transferred into buffer A [50 mM Tris/HCl and 1 mM EDTA (pH 7.2)] and passed down a Sephacryl S-200 SEC column (GE Healthcare; 26 × 60 cm; AKTA purifier system). Pure BM3 fractions (checked by SDS/PAGE) were concentrated by ultrafiltration (Vivaspin sample concentrator with molecular mass cut-off at 30 kDa; Millipore) and stored in buffer A plus 50 % (v/v) glycerol at − 80 ◦ C. For nontagged BM3 haem domains, a further 30–60 % ammonium sulfate cut was applied. The P450-containing pellet was resuspended in buffer A and dialysed into the same buffer to desalt, then further purified by anion-exchange chromatography on an AKTA purifier system using a Q-Sepharose anion-exchange column (16 × 10 cm), with elution in a gradient of 0–500 mM KCl in buffer A. Haem domain fractions were desalted (GE Healthcare column; 26 × 10 cm) on the AKTA into 25 mM KPi (pH 7.0), loaded on to a hydroxyapatite column (Bio-Rad Laboratories; 16 × 11 cm) and eluted in a 200 ml gradient of 25–500 mM KPi (pH 7.0). Pure haem domains were concentrated by ultrafiltration as described above and used immediately for crystallography, or flash-frozen in liquid nitrogen and stored at − 80 ◦ C. As described previously, intact BM3 and haem domain proteins with the A82F mutation were passed through a Lipidex 1000 column (PerkinElmer) in 25 mM KPi (pH 7.0) to remove any fatty acid retained during purification [19]. P450 quantification

Concentrations of the LS (low-spin) forms of WT and mutant intact BM3 enzymes and haem domains were determined using molar absorption coefficients of ε418 = 105 mM − 1 ·cm − 1 and ε 419 = 95 mM − 1 ·cm − 1 respectively, at the Soret maximum [19,20]. Fe(II)CO complexes were formed by bubbling sodium dithionite-reduced WT/mutant BM3 and haem domains (approximately 2–4 μM) with CO gas [21]. WT and mutants showed near-complete formation of the P450 (thiolate-co-ordinated) state, with little of the P420 state (which probably results from cysteine thiol co-ordination) formed in any case [22,23].

Oxidation of proton pump inhibitor drugs

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Fatty acid and PPI drug binding to WT and mutant intact BM3 enzymes

Table 1 Binding affinity and kinetic parameters for the interactions of WT and gatekeeper mutant P450 BM3 enzymes with NPG and PPI substrates

Dissociation constants (K d values) for binding of NPG (Npalmitoylglycine), ESO, LAN, PAN and RAB to WT/mutant intact BM3 enzymes were determined by absorption titrations (∼1–2 μM protein) in 100 mM KPi (pH 7.0; assay buffer) at 25 ◦ C in 1 cm pathlength quartz cuvettes as described previously [19,24]. Titrations were continued until no further P450 haem spectral changes occurred. Difference spectra (produced by subtracting each successive ligand-bound spectrum from that of the ligand-free enzyme) were generated in each titration. Maxima and minima in each set of difference spectra were identified (using the same wavelength pair in each titration) and the overall absorbance changes (Apeak − Atrough ) were plotted against the substrate concentration. Data were fitted using a standard (Michaelis–Menten) hyperbolic function or (where the K d value is 5× the P450 concentration) using the Morrison (quadratic) equation for tight-binding ligands (eqn 1), in order to determine K d values [25,26]. UV–visible spectroscopy was done on a Cary 50 UV–visible spectrophotometer (Agilent). Data analysis and fitting was done using Origin Pro (OriginLab). Amax × (S + E t + K d ) − (((S + E t + K d )2 Aobs = 2E t

Data are shown for binding constants (K d values) and steady-state kinetic turnover of WT, A82F, F87V and AF87V/A82F (DM) intact P450 BM3 enzymes with NPG, ESO, LAN, PAN and RAB. ND indicates that a negligible BM3 haem absorbance shift was observed with selected PPIs (and hence K d values were not determinable), or that insufficient PPI-dependent stimulation of NADPH oxidation (above background) occurred with WT BM3 and certain PPI substrates (ESO, PAN and RAB) to enable determination of accurate kinetic parameters. The tight-binding NPG induces near-complete conversion of the ferric haem iron into the HS form in WT and all mutant BM3 enzymes. Estimates for the percentage spin-state conversion with the PPI drugs are to the nearest 5 %. Data for WT BM3 with NPG are from [19]. Results are means + − S.E.M.

− (4 × S × E t ))0.5 )

(1)

In eqn (1), Aobs is the observed absorbance change at ligand concentration S, Amax is the absorbance change at ligand saturation, Et is the P450 concentration and K d is the dissociation constant for the P450–ligand complex.

BM3 HS haem enzyme Substrate (%) K d (μM) WT A82F F87V DM WT A82F F87V DM WT A82F F87V DM WT A82F F87V DM WT A82F F87V DM

NPG NPG NPG NPG ESO ESO ESO ESO LAN LAN LAN LAN PAN PAN PAN PAN RAB RAB RAB RAB

>95 >95 >95 >95 ND 30 ND 90 ND 55 ND 70 ND 30 ND 50 ND 10 ND 40

0.082 + − 0.011 0.297 + − 0.069 0.204 + − 0.045 0.004 + − 0.003 ND 23.9 + − 3.2 ND 2.89 + − 0.23 ND 140 + − 23 ND 58.6 + − 5.4 ND 25.6 + − 2.7 ND 8.50 + − 0.50 ND 159 + − 15 ND 43.9 + − 2.8

k cat (min − 1 ) K m (μM)

k cat /K m (min − 1 ·μM − 1 )

4770 + − 160 5130 + − 570 4970 + − 240 4050 + − 250 ND 1950 + − 55 1380 + − 20 2090 + − 60 481 + −9 1230 + − 35 1560 + − 45 435 + − 15 ND 2130 + − 110 1410 + − 40 2290 + − 80 ND 1360 + − 35 1110 + − 60 2890 + − 80

343 + − 78 195 + − 61 334 + − 79 2120 + − 475 ND 42.7 + − 4.3 51.5 + − 5.7 641 + − 117 7.8 + − 0.6 38.8 + − 3.3 25.4 + − 2.3 63.5 + − 8.0 ND 41.7 + − 6.8 31.9 + − 3.0 432 + − 56 ND 10.8 + − 1.1 11.0 + − 2.2 83.8 + − 10.1

13.9 + − 2.7 26.3 + − 5.3 14.9 + − 2.8 1.91 + − 0.31 ND 45.7 + − 3.3 26.8 + − 2.6 3.26 + − 0.50 61.8 + − 3.7 31.7 + − 1.8 61.3 + − 3.9 6.85 + − 0.63 ND 51.1 + − 5.7 44.2 + − 2.9 5.30 + − 0.50 ND 126 + −9 101 + − 15 34.5 + − 3.2

Steady-state kinetic analysis of WT and mutant intact BM3 enzymes with fatty acid and PPI substrates

Steady-state kinetic studies were also done on a Cary 50 UV– visible instrument. Substrate (ESO, LAN, PAN and RAB)dependent oxidation of NADPH was determined at 340 nm. BM3 concentration was kept constant (in range 25–150 nM) with the substrate concentration varied and a near-saturating NADPH concentration used (200 μM). Assays were done at 25 ◦ C in assay buffer with a 1 cm pathlength quartz cuvette. Enzyme rate constants for substrate-induced NADPH oxidation were determined in triplicate at each substrate concentration at 340 nm using ε340 = 6.21 mM − 1 ·cm − 1 . Rate constants were plotted against the substrate concentration. Data were fitted to the Michaelis–Menten equation to define the kcat and K m parameters for substrate-dependent NADPH oxidation and are reported in Table 1. EPR spectroscopy

EPR spectroscopy (for ligand-free and drug substrate-bound WT and mutant BM3 enzymes) was performed using a Bruker ELEXSYS E500 EPR spectrometer, operating at X band, fitted with an ESR-900 liquid helium flow cryostat (Oxford Instruments) and a Super High Q (ER-4122SHQE) resonator. Spectra were recorded at 10 K with a microwave power of 0.5 mW and a modulation amplitude of 0.5 mT. Protein samples (200 μM) in KPi buffer (100 mM; pH 7.0) were prepared in (i) the absence of solvent; (ii) with addition of either 1.8 μl of methanol or DMSO solvent; and (iii) with 400 μM PPI drug dissolved in 1.8 μl of solvent. Samples were thus in a final volume of 250 μl in buffer or with buffer plus 0.72 % solvent.

Enzymatic oxidation of substrates and product characterization ESO, LAN, PAN and RAB turnover and analysis by LC–MS

Turnover reactions for oxidation of ESO, LAN, PAN and RAB were done in deep-well blocks at 37 ◦ C with shaking for 30 min. Reaction mixtures contained purified WT or mutant (F87V, A82F or DM) BM3 enzymes (1 μM), substrate (10 μM), NADPH regeneration system (7.76 mM glucose 6-phosphate, 0.6 mM NADP + and 0.75 unit/ml glucose-6-phosphate dehydrogenase) in turnover buffer [50 mM KPi and 5 mM CaCl2 (pH 7.4)] in a final volume of 500 μl. On completion of the reaction, protein was mixed with an equal volume of ACN (acetonitrile) containing 1 μg/ml fluconazole IS (internal standard) by shaking the mixed samples at 800 rev./min for 10 min. Precipitated protein was filtered through protein precipitation plates (Phenomenex) into MS vials (FluidX) and clarified by centrifugation (4000 g for 25 min at 10 ◦ C). Analysis was done on a Thermo Exactive LC– MS with a CTC PAL auto sampler (Thermo Scientific) with a Kinetex 2.6U XB-C18 100 Å column (Phenomenex). A gradient of 0.1 % formic acid to ACN was used to resolve products. Drugs and metabolites were run in positive mode with the molecular ion as M[H] + . All high intensity peaks were selected from the total ion chromatogram and analysed using Thermo Xcalibur quantification software, along with the fluconazole IS. This software then gave total ion readings for both the IS and the metabolites formed. The total ion data were then corrected for the IS and for any degradation of product that occurs nonenzymatically. Additional MS fragmentation analysis for PAN was carried out on an Agilent 6550 iFunnel Q-TOF LC–MS with 1290 Infinity LC system. A ZORBAX Eclipse Plus C18 c The Authors Journal compilation c 2014 Biochemical Society

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(2.1 × 50 mm; 1.8 μm) Rapid Resolution HT column (Agilent) was used with a gradient of 0.1 % formic acid to ACN to resolve products. Turnover reactions with the DM BM3 were done as described above, but in single vials with total reaction volumes of 5 ml. Products were extracted using Strata-X SPE columns (Phenomenex), dried under vacuum and eluted in 50:50 ACN/methanol. Fragmentation data analysis was performed with the MassHunter MSC (Molecular Structure Correlator) program (Agilent). ESO, LAN and RAB turnover and analysis by NMR

Turnover reactions with ESO, LAN and RAB were done in 100– 500 ml flasks at 37 ◦ C with shaking of reagents at 100 rev./min for 2 h. Reaction mixtures contained purified WT or mutant (F87V, A82F or DM) intact BM3 enzymes (1 μM), substrate (10–100 μM), and the NADPH regeneration system in 60–500 ml of assay buffer. Products were extracted using Strata-X SPE columns, dried under vacuum and eluted in 50:50 ACN/methanol, followed by drying under nitrogen and water removal by freeze drying. Analysis was done on a Bruker Avance 400 MHz NMR. 1 H spectra were collected at 400 MHz and 13 C spectra at 101 MHz. Spectra were baseline corrected and referenced to TMS (tetramethylsilane) standard by the residual non-deuterated solvent in the sample. δ values are given in p.p.m. and J values are in Hz. Full assignments were made by COSY, HMBC (heteronuclear multiple bond correlation) and HMQC methods. Signal splittings were recorded as singlet (s), doublet (d), doublet of doublets (dd) αβ system (AB) and multiplet (m). Processing was carried out using MestReNova Lite (Mestrelab Research, Santiago de Compostela, Spain) and ACD NMR Processor (Advanced Chemistry Development). Crystallization of the DM BM3 haem domain and its ESO-bound complex and determination of its structure

Crystallography was performed using the sitting-drop method using a seeding protocol at 4 ◦ C. Crystals obtained for the DM haem domain during initial screens were used to create microcrystal screen stocks and consecutive screens (Molecular Dimensions) were made with drops that consisted of 150 nl of the DM haem domain protein (230 μM), 50 nl of seed stock and 200 nl of well solution using a Mosquito liquid handling robot (TTP LabTech). For the ESO–DM haem domain complex, the protein was saturated with ESO ligand before crystallization. ESO was titrated into the DM haem domain until no further change in haem iron spin state towards HS (high spin) was observed. Thereafter samples were concentrated by ultrafiltration in the presence of saturating ESO. Microseeding was also used to produce diffraction quality crystals. Crystals were obtained under a range of conditions and flash-cooled in liquid nitrogen before data collection. The mother liquor was supplemented with 10 % PEG 200 where an additional cryoprotectant was required. Data were collected at Diamond synchrotron beamline IO2 (Harwell, U.K.) and were reduced and scaled using XDS [27]. Structures were solved by molecular replacement with the previously solved BM3 haem domain structure in complex with OMP (PDB code 4KEY) using PHASER [28]. Structures were refined using REFMAC5 [29] and Coot [30]. Materials

Oligonucleotide primers for mutagenesis were from Eurofins MWG Operon. ESO and LAN were from Sigma–Aldrich, c The Authors Journal compilation c 2014 Biochemical Society

and PAN, RAB and all standards were from Santa Cruz Biotechnology. Bacterial growth medium (TB) was from Melford. Unless specified, other chemicals were from Sigma–Aldrich and of the highest purity available. RESULTS UV–visible spectroscopic binding studies of BM3 ‘gatekeeper’ mutants with diverse PPI drugs

In an previous study, we were able to show that the fatty acid hydroxylase P450 BM3 underwent a conversion of specificity into an OMP-binding/oxidizing P450 on introduction of one or both of the A82F and F87V mutations in the P450 active-site channel [19]. Structural studies showed that the A82F mutation altered the conformational status of the BM3 haem domain to facilitate OMP binding, whereas the F87V mutation improved OMP access to the haem active site for effective catalysis [19]. To investigate whether these mutations also facilitated binding of other PPI drugs in clinical use, we examined P450 haem spectral perturbation with a series of other PPI drugs of diverse structures (Figure 1). We selected the major PPI drugs ESO (the pharmacologically active Senantiomer of OMP), LAN, PAN and RAB. P450 spectral binding studies were done to ascertain whether these PPIs could bind to mutant (and WT) BM3 P450s, and if binding induced a LS to HS shift in the ferric haem iron spin-state equilibrium that is typical of P450 substrate association. This is usually observed as a haem Soret band maximum shift from ∼418 to ∼392 nm when fatty acids bind to BM3. Optical binding studies were done with each PPI and the WT, A82F, F87V and F87V/A82F (DM) intact P450 BM3 proteins, and binding data compared with those collected previously with NPG, a tight-binding fatty acid-derived substrate for BM3 [19,31]. Control studies showed no discernible haem spectral perturbation caused by the solvents (DMSO and methanol) in absence of PPIs. NPG gives a near-complete haem iron HS shift for WT and all mutants, with K d values less than 1 μM (Table 1). However, the binding studies with the selected PPIs showed no spectral perturbation with WT BM3, suggesting negligible productive binding. The F87V mutant also showed no discernible spectral shifts upon titration with the PPIs. However, both the A82F and DM BM3 mutants bound all four PPIs with substantial HS shifts, thus indicating that the A82F mutation is a primary determinant of altered selectivity and productive binding mode for these drugs. In each case, there was an additive effect on PPI affinity by inclusion of the F87V mutation, with the DM showing a greater proportion of HS shift than the A82F mutant and with lower PPI K d values for the DM BM3 in each case. This is probably due to the influence of the F87V mutation in increasing the size of the active-site cavity and allowing the drugs to move further towards the haem. ESO shows the highest affinity, with an A82F K d value of 23.9 μM and a DM K d value of 2.89 μM, associated with a near-complete HS conversion. LAN gives an ∼70 % shift to the HS form in the DM BM3, with K d values of 140 μM for A82F and 58.6 μM for the DM. PAN gives up to an ∼50 % shift with the DM, and with an A82F K d value of 25.6 μM and DM K d value of 8.5 μM. RAB showed relatively little HS shift with A82F (∼10 %) and comparatively weak binding (K d = 159 μM); however, RAB binding was greatly improved in the DM with ∼40 % HS accumulated at saturation and a K d value of 43.9 μM (Table 1). Binding spectra for the DM BM3 enzyme with each of the PPI drugs are shown in Figure 2. These UV–visible spectroscopic data indicate that diverse PPI class compounds bind the A82F mutation-containing BM3 gatekeeper mutants. Binding occurs in proximity to the haem catalytic site to displace the haem iron’s distal (sixth) water ligand, and thus the


Figure 1

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PPI drug structure and functional groups

Backbone PPI structure is shown with variations at the R groups for each compound indicated in the Table.

PPIs occupy a productive mode similar to that for the fatty acid substrates of P450 BM3. Steady-state kinetic analysis of PPI turnover by BM3 mutants

Steady-state kinetic studies (in presence of near-saturating NADPH) were performed on intact WT and BM3 mutants, using each of the PPIs at a range of substrate concentrations and comparing the kinetic data with those with NPG as the substrate [19]. As shown in Table 1, the efficient NPG lipid substrate has high kcat values (4050–5130 min − 1 ) and low K m values (26.3–1.9 μM) with WT and the F87V, A82F and DM BM3 enzymes, leading to high catalytic efficiency in all cases. With WT BM3, only LAN showed significant substrate-stimulated NADPH oxidation over background levels (kcat = 481 min − 1 ), with a LAN K m value of 61.8 μM. In contrast with the data for the WT BM3, all of the BM3 mutants showed substrate-stimulated oxidation with the four PPIs. DM BM3 showed greatest affinity (lowest K m values) for each of the PPIs, and the highest (for RAB) or joint highest activity, within the margin of error, along with A82F for ESO and PAN (Table 1). However, F87V BM3 proved the fastest LAN-dependent NADPH oxidase (kcat = 1560 min − 1 ) (Figure 3). Indeed, despite little evidence for PPI-dependent HS conversion of its haem iron, the F87V BM3 kcat values for PPI-dependent NADPH oxidation were >1000 min − 1 in all cases. The superior PPI-binding affinity exhibited by DM BM3 in its K m values (and mirrored in the K d values) underpins DM BM3 having the highest catalytic efficiency (kcat /K m ratio) for PPI-dependent NADPH oxidation in all cases. DM BM3 also exhibits enhanced binding to the NPG lipid substrate compared with WT BM3 (K m values of 1.91 and 13.9 μM respectively; Table 1). EPR spectroscopy

The EPR data for the WT P450 BM3 haem domain (Figure 4, A), show a rhombic signal with a single set of g values at gz = 2.41, gy = 2.25 and gx = 1.92 (2.41/2.25/1.92). These are consistent with previously reported data for WT BM3 (2.42/2.25/1.92) and indicative of a LS ferric haem with axial co-ordination to the central iron provided by cysteine thiolate and water [32]. There is no significant signal corresponding to a HS ferric state (results not shown). Addition of the solvent DMSO (at final concentration

0.72 %) perturbs the EPR spectrum (Figure 4, B) and gives rise to a new set of LS ferric haem g values at 2.45/2.25/1.91.These data indicate that DMSO solvent affects the environment around the haem iron, probably influencing the locations of water molecules within the active site. Addition of PPI substrates did not produce any significant formation of HS haem iron in the WT and mutant BM3 EPR spectra (results not shown). WT BM3 exhibits a new species in the LAN-bound form that is different from that induced by DMSO alone (Figure 4, C). The g values for this new LS species are at 2.40/2.25/1.92. A very similar EPR spectrum was observed for WT BM3 bound to RAB (results not shown). The F87V mutation has a significant effect on the EPR spectrum with the substrate-free enzyme showing new LS species (Figure 4, D). Three LS states are observed in the native form (2.48/2.25/1.87, 2.45/2.25/1.90 and 2.41/2.25/1.92), and these are almost unchanged on addition of methanol solvent at 0.72 % (2.48/2.25/1.90, 2.43/2.25/1.91 and 2.40/2.25/1.92; results not shown). Thus the organization of water molecules in the haem iron environment is clearly perturbed in the F87V mutant, as a consequence of the removal of the aromatic residue in the vicinity of the haem iron. However, the F87V EPR spectrum is converted into only one major species after the addition of DMSO (2.44/2.25/1.91; Figure 4, E). PPI addition does not then lead to further major changes in the EPR spectra. The A82F haem domain also shows three LS states in absence of solvent/substrate (2.47/2.25/1.90, 2.42/2.25/1.92 and 2.39/2.25/1.92; results not shown), again indicative that this mutation perturbs active-site water organization around the haem iron. Addition of solvent reduces the signal complexity (2.44/2.25/1.91 for DMSO; 2.44/2.25/1.90 and 2.42/2.25/1.91 for methanol; results not shown), but addition of PPI drugs again has little further influence on the EPR spectra. The native BM3 DM haem domain has an even more complex LS EPR spectrum (2.53/2.25/1.87, 2.49/2.25/1.90, 2.44/2.25/1.91 and 2.41/2.25/1.91; Figure 4, F) indicative of the combined influence of the A82F and F87V mutations on the distal haem co-ordination environment. The spectral complexity is decreased slightly on addition of methanol/DMSO solvents, but binding of the PPI drugs again has only marginal influence on the EPR spectrum above that induced by solvent alone, e.g. g values of 2.46/2.25/1.90 and 2.43/2.25/1.91 for the ESO-bound DM haem domain (Figure 4, G). Collectively, these EPR data demonstrate clearly that major alterations in the environment around the iron distal pocket c The Authors Journal compilation c 2014 Biochemical Society

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Figure 2


Binding of diverse PPI drugs to the P450 DM BM3 enzyme

(A) UV–visible spectra for F87V/A82F (DM) intact BM3 mutant (∼1.1 μM) titration with ESO, with the most intense spectrum at ∼418 nm being that for the ligand-free LS form. Other selected spectra shown are shown at ESO concentrations of 0.9, 1.8, 5.4 and 36 μM, showing progressive spectral shift to a new HS species at ∼392 nm. The inset shows a plot of the Soret absorption change (A389 –A421 ) against the [ESO], with data fitted using eqn (1) to yield a K d = 2.89 + − 0.23 μM. (B) Comparable data set for the binding of LAN to DM BM3 (0.85 μM) with LAN-bound spectra shown at 10, 20, 30, 50 and 70 μM substrate concentrations. The inset shows plot of (A389 –A421 ) against the [LAN], fitted as previously to give K d = 58.6 + − 5.4 μM. (C) The data set for PAN binding to the DM (0.85 μM) with substrate-bound spectra at 3.75, 10, 20 and 35 μM PAN. The inset shows plot of (A389 –A421 ) against [PAN], fitted as previously to give a K d = 8.5 + − 0.5 μM. (D) The data set for RAB binding to the DM (1.5 μM) with RAB-bound spectra shown at 10, 30, 40, 80 and 200 μM substrate concentrations. Inset shows plot of (A388 –A421 ) against the [RAB], fitted to give a K d = 43.9 + − 2.8 μM.

are induced by F87V, A82F and F87V/A82F mutations in the respective haem domains. DMSO influences the distal water environment considerably in WT and all gatekeeper BM3 mutants, but the effects of methanol are less profound. However, the addition of PPI drugs to the BM3 mutants (despite them inducing substantial HS shifts in the haem iron spin-state equilibrium at ambient temperature for the A82F and DM enzymes) causes only very minor further changes to the EPR spectra at 10 K.

Oxidative turnover of PPI drugs by WT and mutant BM3 enzymes using LC–MS

To analyse the catalytic activity of the WT and mutant forms of BM3, in vitro turnover of each PPI was performed with intact WT, F87V, A82F and DM BM3 enzymes, and products were analysed by LC–MS. Oxidation of each of the drugs was observed, as detailed below.

ESO

The LC–MS analysis for the ESO substrate showed a M[H + ] of 346.1224 for the starting material, with a natural fragmentation occurring between the sulfoxide sulfur and the methoxybenzimi c The Authors Journal compilation c 2014 Biochemical Society

-dazole moiety to give the corresponding 198.0587 species. Upon enzymatic turnover, a + 16 increase in these m/z peaks to 362.1134 and 214.0536 was observed, indicating insertion of oxygen into the pyridinyl fragment, generating oxidized ESO. Data for ESO oxidation by the DM BM3 enzyme are shown in Supplementary Figure S1 (http://www.biochemj.org/bj/ 460/bj4600247add.htm). The product quantities for ESO oxidation were high, with ∼90 % of a single monohydroxylated product obtained for both the F87V and DM BM3 enzymes. The A82F BM3 enzyme produced ∼40 % of the same monohydroxylated product in the same time (Figure 5A). It is interesting to note that, in contrast with previous studies using OMP [19], the oxidation of ESO results in negligible (95 %, with >65 % dealkylated thioether, >15 % demethylated thioether and ∼15 % demethylated RAB (Figure 5D). The major human metabolites of RAB are the non-enzymatically produced thioether, and the CYP-mediated metabolites being the demethylated RAB and the RAB sulfone, produced by CYP2C19 and CYP3A4 respectively [35–37].

Oxidation of PPIs by WT and mutant BM3 enzymes using NMR

NMR spectroscopy was used for the analysis of the substrates and the main oxidized products from ESO, LAN and RAB turnover by WT and gatekeeper BM3 mutants. Large-scale enzymatic turnover reactions were carried out for each substrate in order to enable use of NMR for identification of the products seen in the LC–MS analysis. Confirmation of the position of oxidation of ESO at the 5-methyl group was obtained from a combination of 1 H, 13 C and 2D NMR (Supplementary Figures S5–S8 at http://www.biochemj.org/bj/460/bj4600247add.htm). Confirmation of the position of oxidation of ESO at the 5-methyl group (as catalysed by human CYP2C19 [38]) was obtained from a combination of 1 H, 13 C and 2D NMR. Analysis of a LAN standard along with metabolites showed the sulfone to be the product (formed when a second oxygen atom is introduced to the central sulfur atom) (Supplementary Figures S9–S11 at http://www.biochemj.org/bj/460/bj4600247add.htm). The LAN sulfone is also the major human metabolite formed by the human CYP3A4 [39]. COSY and HMBC spectra were used to make full assignments of LAN and the LAN sulfone. NMR studies of RAB metabolism by the BM3 DM enzyme provided evidence for RAB substrate inhibition, since the proportions of products formed at higher RAB concentrations (50 μM and 100 μM) differed from those at 10 μM RAB (as also determined by LC–MS analysis). The major product formed at 100 μM RAB was the RAB thioether, whereas at 50 μM RAB the demethylated RAB was most prevalent. LC–MS analysis provided evidence for its further oxidative breakdown to the dealkylated thioether (results not shown), which is the major product observed at 10 μM RAB (Figure 5D). Full NMR spectral assignments in each case are given in the Supplementary Online Data (http://www.biochemj.org/bj/460/bj4600247add.htm). Assignments for RAB and its oxidation products were


Figure 5

255

Proportions of PPI turnover products identified by LC–MS

The proportions of products from different PPI drugs seen from LC–MS analysis after a 30 min incubation with WT and mutant BM3 enzymes. Data were corrected for an IS (fluconazole), and reflect data averages of two repeats with error bars showing S.E.M. WT (black), A82F (white), F87V (grey) and DM (dashed lines). (A) Esomeprazole showing proportions of ESO, 5-hydroxy ESO (ESO-OH) and 5-carboxy ESO (ESO-COOH). (B) Lansoprazole showing proportions of LAN, LAN sulfone (LAN SUL) and hydroxylated LAN (LAN-OH). (C) Pantoprazole showing proportions of PAN and PAN N -oxide (PAN N-OX). (D) Rabeprazole showing proportions of RAB, dealkylated RAB thioether (RAB TE DA), demethylated RAB (RAB DM) and demethylated RAB thioether (RAB TE DM).

made by 1 H, COSY and HMBC NMR (Supplementary Figures S12–S16 at http://www.biochemj.org/bj/460/bj4600247add. htm). X-ray crystallography

The structure of ESO bound to the DM BM3 haem domain was solved to 1.83 Å resolution (PDB code 4O4P), using molecular replacement with our previously determined DM haem domain OMP-bound structure (PDB code 4KEY) (Supplementary Table S1 http://www.biochemj.org/bj/460/bj4600247add.htm) [19]. As observed for the OMP complex, the ESO-bound structure is highly similar to the WT fatty acid complex [41], despite the dissimilarity in ligand nature. An overlay of the DM ESO-bound active-site ligand density with the OMP-bound DM structure (PDB code 4KEY) is shown in Figure 6. The ESO-bound DM structure reveals that the sulfoxide oxygen is lost in the PPI, either due to synchrotron X-ray irradiation or, more probably, from the breakdown of ESO in the aqueous environment [40]. Thus, in both DM haem domain structures, OMP/ESO are bound as the non-chiral thioether forms. This prevents us from gaining a clear structural rationale as to why OMP binds more tightly to DM BM3 than does ESO (OMP K d = 0.212 μM compared with 2.89 μM for ESO) [19], but yet is metabolized less selectively than ESO.

Figure 6 Overview of the S -omeprazole (ESO)-binding pocket in the DM BM3 haem domain structure Residues in contact with the ESO ligand are shown in atom coloured sticks (blue carbons). ESO is shown with yellow carbons, whereas the haem cofactor is shown with purple carbons. Water molecules in close contact with the ligand are shown as spheres. The 2F o −F c omit map density corresponding to the ligand is shown as a green mesh, contoured at 3σ .

DISCUSSION

Production of drug metabolites is of interest to the biocatalysis industry as these compounds are often expensive and difficult to produce using conventional chemical methods [1]. Most human drug metabolites are formed directly or indirectly by P450 enzymes. Understanding the pharmacology and toxicology of

these metabolites is crucial to develop a full understanding of the mode of action of the parent drug, and to identify toxicity issues. The FDA requires that drug metabolites formed to significant levels in vivo are subjected to similar rigorous testing as the c The Authors Journal compilation c 2014 Biochemical Society

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Figure 7


Reactions schemes outlining pathways of P450 metabolism of PPI drugs

P450 BM3 pathways are shown with the relevant human P450 catalysts named.

parent drug [42]. However, production of sufficient amounts of drug metabolites for testing is challenging, particularly where regio- and/or stereo-selective drug oxidation by P450s occurs. Recombinant human P450s may generate small amounts of metabolites, but this may be impractical at a large scale due to poor enzyme stability, slow rates and requirement for an exogenous (CPR) redox partner. In contrast, P450 BM3 has the highest rates of substrate oxidation across the P450 superfamily and is a soluble catalytically self-sufficient enzyme. BM3 has been a test bed for P450 mutagenesis, and its activity profile has been substantially altered by protein engineering approaches [18,43]. Our previous studies showed that single mutations in the BM3 haem domain dramatically alter substrate selectivity to enable binding and oxidation of the PPI omeprazole, with the gatekeeper A82F mutation (in particular) altering conformational stability of BM3 to facilitate its diversification of substrate selectivity [19]. In the present study, we report the binding and oxidation of four other members of the PPI drug class by WT, A82F, F87V and F87V/A82F (DM) BM3 enzymes. ESO, LAN, PAN and RAB all bind to and/or are oxidized by the BM3 gatekeeper mutants. c The Authors Journal compilation c 2014 Biochemical Society

PPI binding induces a substrate-like type I haem absorbance shift for the A82F and DM BM3 enzymes, indicating a change in the P450 ferric haem iron equilibrium from LS towards HS. The F87V mutation improves PPI binding in each case, with K d values consistently being lower for DM BM3 than for the A82F BM3 point mutant (Table 1). EPR spectroscopy reveals (through differences in the LS haem signal) major differences in the distal environment of the haem iron in the BM3 mutants compared with the WT enzyme. These probably reflect altered water environments around the sixth co-ordination position of the iron that arise as a consequence of structural changes in the immediate haem environment (induced by the F87V mutation) and through altered conformational dynamics in the haem domain (in the A82F and DM enzymes). It is important to note that the addition of DMSO as the carrier for certain PPI drugs also influences the BM3 LS EPR spectrum, again probably influencing the solvation state around the distal haem iron site (Figure 4). X-ray crystallographic studies of WT and F87A mutants of the BM3 haem domain indicated that co-crystallization of the mutant with DMSO at 28 % (v/v) resulted in a direct interaction of DMSO with the haem iron. However, the DMSO concentration


used for EPR in the present study is substantially lower (0.72 %) [44]. Despite limited PPI-induced HS haem development, the F87V BM3 enzyme proved an effective catalyst for oxidation of each of the PPI drugs, albeit with lower catalytic efficiency than DM BM3 (Table 1). Steady-state kinetic analysis of the BM3 gatekeeper mutants indicated PPI substrate-dependent NADPH oxidation at rates up to ∼50 % of those for turnover of the lipid substrate NPG. However, PPI product formation was more extensive for ESO and RAB (and for the racemate OMP) than for LAN and PAN, indicating that electron transfer was less efficiently coupled to substrate oxidation for LAN/PAN [19]. The altered conformational flexibility of the A82F-containing BM3 enzymes underpins their ability to bind the different PPI drugs, with the F87V mutation altering accessibility to the haem active site and playing an important function in enhancing enzymatic efficiency in most cases. However, the F87V mutation alone is sufficient to facilitate substantial improvements in oxidation of certain PPIs, and particularly in the case of ESO, where product formed in unit time is similar to that from DM BM3. Although structural similarity in the PPI drug ‘core’ (Figure 1) in part explains the ability of diverse PPIs to bind the different BM3 gatekeeper mutants, altered modes of binding for the tested PPIs is also evident from the different positions at which oxidation occurs on these molecules (Figure 5). Importantly, our data reveal that in many cases the PPI products formed by BM3 mutants replicate the major metabolites formed by their human P450 counterparts with the same drugs (Figure 7). More specifically, ESO is transformed by BM3 (A82F, F87V and DM) to 5-OH ESO, which is the major human metabolite produced by CYP2C19 [45]. The data for ESO differ from those for the oxidation of the racemate OMP by the BM3 mutants, since there is negligible formation of the 5-COOH ESO product. These data suggest that the primary 5-OH ESO (5-OH S-OMP) metabolite does not bind productively to the BM3 variants, whereas the 5-OH R-OMP component from the racemate may be able to do so to enable formation of the 5-COOH OMP product. These results further suggest that, as with the human P450s, OMP metabolism is specific for each enantiomer [33]. The major metabolites seen in humans for LAN are the sulfone and the 5-OH LAN, which are produced by CYP3A4 and CYP2C19 respectively [46]. Our data show the primary product with our mutant BM3 enzymes (with up to 33 % conversion by the DM) is the LAN sulfone. In the case of PAN, the extent of product formation is the lowest among the PPIs tested, but the product is exclusively the PAN N-oxide. In this case, the enzymatic reaction (catalysed most effectively by the DM BM3) produces a metabolite that is also observed in lower amounts as a by-product of PAN synthesis. The major pathway for RAB metabolism is its non-enzymatic (reductive) conversion into the thioether, with up to 50 % conversion in aqueous solution in 1 h [47,48]. This product is also observed in the present study, and control reactions showed that BM3 enzymes alone had little effect on the extent of this reaction, but that the inclusion of an NADPH cofactor regeneration system approximately doubled the amount of thioether product formed, probably due to providing a more reducing environment for the reaction. The other major human P450 metabolites of RAB are the demethylated and sulfone forms, as produced by CYP2C19 and CYP3A4 respectively [48]. Using the BM3 DM enzyme, the RAB product profile showed dependence on substrate concentration. At 100 μM RAB the thioether was the major product observed, whereas lowering the RAB concentration favoured the demethylated RAB (at 50 μM) or a novel metabolite involving the loss of the entire ether chain,

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producing a dealkylated thioether of RAB (the demethoxypropyl RAB thioether at 10 μM RAB). Substrate inhibition and substrate concentration dependency on product outcome is frequently seen in human P450 reactions, e.g. in the case of CYP2C9-catalysed methyl-hydroxylation of the non-steroidal anti-inflammatory drug celecoxib and for the CYP2D6catalysed O-demethylation of the antitussive dextromethorphan [49]. In conclusion, the results of the present study demonstrate that relatively limited mutagenesis of P450 BM3 can generate considerable diversity in substrate selectivity, with the utility of the BM3 gatekeeper mutants A82F, F87V and A82F/F87V now expanded to their oxidation of a range of PPI drugs in clinical use. More importantly, in most cases the products formed from these PPIs are the same as those generated by the major human drug-metabolizing P450s, notably CYP3A4 and CYP2C19. By lowering the energetic barrier to conformational reorganization and transition to a substrate-bound state, the mutations facilitate recognition of diverse PPI substrates that are disfavoured by WT BM3. The diversity in substrate recognition and oxidation among the major human hepatic drug-metabolizing P450s is considered crucial for the ability of a small number of such enzymes to metabolize and detoxify a wide range of xenobiotics [50]. To achieve this end, evolutionary pressure probably selected conformationally flexible P450 variants able to accommodate molecules of diverse size and chemical character. The ability to radically change the substrate selectivity of P450 BM3 with point mutations that are structurally destabilizing in addition to altering the active site environment thus points to routes by which the hepatic P450s may also have evolved efficiently to deal with a plethora of environmental toxins.

AUTHOR CONTRIBUTION Christopher Butler, Caroline Peet, Kirsty McLean, David Leys, Michael Voice and Andrew Munro conceived and designed the experiments. Christopher Butler, Caroline Peet, Kirsty McLean, Richard Blankley, Karl Fisher, Stephen Rigby, David Leys and Michael Voice performed the experiments. Christopher Butler, Caroline Peet, Kirsty McLean, Michael Baynham, Richard Blankley, Stephen Rigby, David Leys, Michael Voice and Andrew Munro analysed the data. Christopher Butler and Andrew Munro wrote the paper. Kirsty McLean, Stephen Rigby and David Leys contributed to writing the paper.

ACKNOWLEDGEMENTS We thank Dr Robert Sˇ ardz´ık for helpful discussions on NMR analysis, and Dr Colin Levy for assistance with synchrotron X-ray data collection.

FUNDING The work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/K001884/1 (to A.W.M./D.L.)] and an Industrial CASE studentship (BB/G01698/1) with Cypex Ltd to A.W.M./M.W.V., supporting C.F.B.

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Received 7 January 2014/3 March 2014; accepted 4 March 2014 Published as BJ Immediate Publication 4 March 2014, doi:10.1042/BJ20140030

c The Authors Journal compilation c 2014 Biochemical Society

Biochem. J. (2014) 460, 247–259 (Printed in Great Britain)

doi:10.1042/BJ20140030

SUPPLEMENTARY ONLINE DATA

Human P450-like oxidation of diverse proton pump inhibitor drugs by ‘gatekeeper’ mutants of flavocytochrome P450 BM3 Christopher F. BUTLER*, Caroline PEET†, Kirsty J. MCLEAN*, Michael T. BAYNHAM‡, Richard T. BLANKLEY‡, Karl FISHER*, Stephen E. J. RIGBY*, David LEYS*, Michael W. VOICE† and Andrew W. MUNRO*1 *Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. †Cypex Ltd, 6 Tom McDonald Avenue, Dundee DD2 1NH, U.K. ‡Agilent Technologies UK Ltd, Lakeside, Cheadle Royal Business Park, Stockport, Cheshire SK8 3GR, U.K.

RESULTS

Figures S1–S16 (as described in the main text) are on the following pages. These provide data from LC–MS analysis (Figures S1–S4) characterizing the oxidation of the PPI drugs ESO, LAN, PAN and RAB by gatekeeper (F87V, A82F, and F87V/A82F) mutants of flavocytochrome P450 BM3. In addition, NMR spectroscopy was used for the analysis of the substrates and the main oxidized products from ESO, LAN and RAB turnover (Figures S5–S16). Table S1 follows Figures S1–S16 and contains crystallographic data for the ESO complex of the P450 BM3 DM (F87V/A82F) haem domain. NMR analysis of ESO and its metabolite from oxidation by BM3 gatekeeper mutants

Confirmation of the position of oxidation of ESO at the 5-methyl group was obtained from a combination of 1 H, 13 C and 2D NMR. ESO starting material was assigned as: 1 H NMR (CDCl3 400 MHz) δ = 8.13 (S, 1H), 7.46 (d, J = 8.84), 6.95 (Broad S, 1H), 6.87 (dd, J = 8.97, 2.40 Hz, 1H), 4.69 (AB, J = 13.52, 11.12 Hz, 2H), 3.77 (S, 3H), 3.63 (S, 3H), 2.16 (S, 3H), 2.15 (S, 3H). 13 C NMR (CDCl3 101 MHz) δ = 164.5, 149.7, 148.7, 127.1, 126.5, 60.8, 60.0, 55.8, 13.4, 11.6. LC–MS [M + H] + 346.1224 (C17 H20 N3 O3 S). As can be seen from the ESO NMR spectra (Figures S5–S8), the product peaks show generation of a single new methyl singlet at 2.14, slight shifts in each methoxy group, a new singlet δ 4.69 corresponding to a methoxy group and a shift in the pyridinyl hydrogen from δ 8.13 to δ 8.37. This pattern is indicative of hydroxylation at one of the pyridinyl methyl groups to form a methoxy group. The coupling seen in the 2D NMR combined with the downfield shift of the pyridinyl hydrogen signal and the lack of shift in the AB system indicates hydroxylation at the 5-position, the same reaction as that catalysed by the human CYP2C19. Upon enzymatic oxidation of ESO, a mixture of starting material and monohydroxylated product was identified. The product was assigned to 5-OH ESO and the additional peaks seen were: 1 H NMR (CdCl3 400 MHz) δ = 8.37 (S, 1H), 7.56 (Broad S, 1H), 6.96 (d, J = 2.40 Hz, 1H), 6.93 (dd, J = 8.84, 2.40 Hz, 1H), 4.76 (AB, J = 13.64, 11.65 Hz, 2H), 4.69 (S, 2H), 3.82 (S, 3H), 3.67 (S, 3H), 2.14 (S, 3H). 13 C NMR (CDCl3 101 MHz) δ = 164.5, 149.6, 148.2, 127.1, 126.6, 61.2, 58.4, 55.8, 13.4, 11.6. LC–MS [M + H] + 362.1134 (C17 H20 N3 O4 S).

NMR analysis of LAN and its major metabolite from oxidation by BM3 gatekeeper mutants

NMR analysis of LAN product along with the LAN standard showed the sulfone to be the major product formed. This occurs by addition of a second oxygen atom to the central sulfur. The LAN sulfone is also the major human metabolite formed by the human CYP3A4. COSY and HMBC spectra were used to make full assignments of LAN and the LAN sulfone. As can be seen from the NMR spectra for the LAN standard and the LAN turnover reaction (Figures S9–S11), there is a mixture of the LAN starting material and the product LAN sulfone following turnover. The combination of LC–MS and NMR analysis showed that, although there was a + 16 increase in the product, there was no discernible hydroxylation on a LAN carbon. The only change observed was the loss of the AB system quartet to give a singlet at δ 5.09. The peak positions and coupling pattern in this product was the same as that seen in the LAN sulfone standard (Santa Cruz Biotechnology), giving us conclusive proof that our product is the sulfone. LAN assignment: [DMSOd6 (deuterated DMSO) 400 MHz] δ 13.59 (S, 1H), 8.29 (d, 1H, J = 5.68 Hz), 7.69 (Broad d, 2H), 7.31 (Broad d, 2H), 7.09 (d, 1H, J = 5.68 Hz), 4.91 (q, 2H, J = 8.72 Hz), 4.80 (AB, 2H, J = 13.77 Hz), 2.18 (S, 3H). 13 C NMR (DMSOd6 101 MHz) 161.25, 154.14, 150.90, 148.10, 125.13, 122.37, 122.02, 107.00, 64.59 (q), 59.94, 10.52. LAN sulfone standard: (DMSO d6 400 MHz) δ 13.79 (Broad S, 1H), 8.11 (d, 1H, J = 5.68 Hz), 7.69 (Broad S, 2H), 7.39 (m, 2H), 7.07 (d, 2H, J = 5.68 Hz), 5.12 (S, 2H), 4.91 (q, 2H, J = 8.72 Hz), 2.23 (S, 3H). 13 C NMR (DMSOd6 101 MHz) 161.43, 148.07, 147.86, 147.76, 123.26, 122.35, 107.47, 64.78, 64.44, 60.37, 10.91. DM BM3 LAN turnover product: (DMSOd6 400 MHz) δ 13.62 (Broad S, 1H), 8.13 (d, 1H, J = 5.68 Hz), 7.66 (Broad S, 2H), 7.35 (m, 2H), 7.09 (app. t, 1H, J = 5.68 Hz), 5.09 (S, 2H), 4.91 (q, 2H, J = 8.72 Hz), 2.22 (S, 3H). NMR analysis of RAB and its metabolites from oxidation by BM3 gatekeeper mutants

As indicated in the main text, the proportions of the different products formed by BM3 gatekeeper mutants showed dependence on the concentration of the RAB substrate used. The main products observed were: (i) the thioether at 100 μM substrate; (ii) the demethylated thioether at 50 μM substrate; and (iii) the dealkylated thioether at 10 μM substrate. Product identification

1 To whom correspondence should be addressed (email [email protected]). The structural co-ordinates reported for ESO bound to the DM BM3 haem domain have been deposited in the PDB under code 4O4P.


C.F. Butler and others

was achieved by a combination of LC–MS and NMR analysis, with LC–MS analysis providing the key evidence for the final oxidative conversion to the dealkylated thioether at 10 μM RAB. Assignments for RAB and for its oxidation products at 50 μM and 100 μM RAB were made using 1 H, COSY and HMBC NMR, and assessments of missing peaks were made with reference to the 1 H product spectra (Figures S12–S16). At the highest RAB substrate concentration (100 μM), the product formed (using DM BM3) is primarily the RAB thioether, as shown by loss of the AB system to form a singlet at δ 4.78 (Figure S15). At the lower RAB concentration (50 μM), NMR analysis shows that the thioether is also formed, but now the singlet at δ 3.26 is also lost. This singlet corresponds to the methyl group on the 3-methoxypropoxy group, showing that a demethylation reaction occurred (Figure S16). LC–MS analysis showed that this was further broken down to the alcohol with loss of the entire 3-methoxypropyl group. RAB standard: (DMSOd6 400 MHz) δ 8.28 (d, 1H, J = 5.56 Hz), 7.46 (m, 2H), 6.93 (d, 1H, J = 5.68 Hz), 6.88 (m, 2H), 4.57 (AB, 2H, J = 12.88 Hz), 4.10 (t, 2H, J = 6.19 Hz), 3.49 (t, 2H, J = 6.32 Hz), 3.25 (S, 3H), 2.17 (S, 3H), 1.98 (quin., 2H, J = 6.19 Hz) 13 C NMR (DMSOd6, 101 MHz) δ 162.61, 152.41, 147.95, 146.62, 121.74, 118.20, 117.32, 105.92, 68.30, 64.92, 59.64, 57.96, 28.67, 10.83. RAB (100 μM) products: 1 H NMR (DMSOd6, 400 MHz) δ 12.63 (Broad S, 1H), 8.23 (d, 1H, J = 5.56 Hz), 7.45 (Broad m, 2H), 7.12 (m, 2H), 6.95 (d, 1H, J = 5.68 Hz), 4.69 (S, 2H), 4.10 (t, 2H, J = 6.19 Hz), 3.48 (t, 2H, J = 6.19 Hz), 3.24 (S, 3H), 2.21 (S, 3H), 1.98 (quin., 2H, J = 6.19 Hz). 13 C NMR (DMSOd6, 101 MHz) 162.64, 154.66, 150.22, 147.76, 119.69, 106.29, 68.26, 65.05, 57.95, 36.22, 28.64, 10.37. RAB (50 μM) products: 1 H NMR (DMSOd6, 400 MHz) δ 12.72 (Broad S, 1H), 8.23 (d, 1H, J = 5.68 Hz), 7.45 (m, 2H), 7.12 (m, 2H), 6.95 (d, 1H, J = 5.81 Hz), 4.69 (S, 2H), 4.12 (t, 2H, 6.19 Hz), 3.58 (t, 2H, J = 6.19), 2.21 (S, 3H), 1.89 (quin., 2H, J = 6.06 Hz). 13 C NMR (DMSOd6, 101 MHz) 162.76, 154.61, 150.25, 147.74, 121.31, 119.68, 106.30, 64.99, 57.04, 36.16, 31.80, 10.37. Table S1 Data reduction and final structural refinement statistics for the ESO (S -omeprazole) complex with the P450 DM BM3 haem domain (PDB code 4O4P) Values in paretheses are for the highest-resolution shell. Parameter

DM–ESO

Space group Cell parameters (A˚) Resolution (A˚) R merge (%) I /σ I R /R free (%) Average B (A˚2 ) RMSD bonds (A˚) RMSD angles (◦ ) Crystallization conditions

P 21 2 1 2 1 a = 59.4, b = 132.9, c = 147.5 66–1.83 (1.88–1.83) 6.7 (61.2) 15.7 (2.5) 18.7/22.1 (30.8/36.4) 22.3 0.025 1.93 20 % PEG4K, 0.2 M MgCl2 and pH 6.5 (0.1 M sodium cacodylate)


Figure S1 LC–MS traces showing ESO and its oxidation by the P450 DM BM3 enzyme (A) Retention time = 5.30 min. Data for ESO before initiation of its oxidation by addition of the DM BM3 enzyme. Peaks at m /z 346.1222 and 198.0587 correspond to fragmentation of ESO at the sulfoxide group (between the sulfur and the methoxybenzimidazole moiety), with the smaller species representing the sulfur-containing fragment. (B) Retention time = 4.92 min. Data following an enzymatic reaction for 30 min. The m /z peaks at 362.1171 and 214.0536 are for the 5-OH ESO and its hydroxylated fragment respectively.


Figure S3 LC–MS traces showing PAN and its oxidation by the P450 DM BM3 enzyme

Figure S2 LC–MS traces showing LAN and its oxidation by the P450 DM BM3 enzyme (A) Retention time = 5.65 min. Data for LAN before initiation of its oxidation by addition of the DM BM3 enzyme. Peaks at m /z 370.0830 and 252.0302 correspond to fragmentation of LAN at the sulfoxide group (between the sulfur and the benzimidazole moiety), with the smaller species representing the sulfur-containing fragment. (B) Retention time = 6.02 min. Data following an enzymatic reaction for 30 min. The m /z peaks at 386.0782 and 163.1330 are for the LAN sulfone product and the benzimidazole moiety of LAN.

(A) Retention time = 5.82 min. Data for PAN before addition of the BM3 DM enzyme and initiation of its oxidation. Peaks at m /z 384.0819 and 200.0375 correspond to fragmentation of PAN at the sulfoxide group (between the sulfur and the benzimidazole moiety), with the smaller species representing the sulfur-containing fragment. (B) Retention time = 6.23 min. Data following an enzymatic reaction for 30 min. The m /z peak at 400.0774 is for the PAN N -oxide, and no fragmentation is seen with this product.



Figure S4

LC–MS traces showing RAB and its oxidation by P450 BM3 mutant enzymes

(A) Retention time = 5.30 min. Data for RAB before addition of BM3 gatekeeper mutant enzymes and initiation of its oxidation. Peaks at m /z 360.1369 and 242.0844 correspond to fragmentation of RAB at the sulfoxide group (between the sulfur and the benzimidazole moiety), with the smaller species representing the sulfur-containing fragment. (B) Retention time = 2.76 min. Data following an enzymatic reaction (with the A82F mutant BM3) for 30 min. The m /z peaks at 346.1220 and 228.0692 are for the demethylated RAB and for its demethylated fragment. (C) Retention time = 3.91 min. Data following an enzymatic reaction for 30 min (with the A82F mutant BM3). The m /z peaks at 330.1269 and 149.0234 are for the demethylated RAB thioether and the benzimidazole thioether fragment. (D) Retention time = 4.20 min. Data following an enzymatic reaction for 30 min (with DM BM3). The m /z peaks at 272.0854 and 149.0235 are for the dealkylated RAB thioether (forming the demethoxypropyl RAB thioether product) and the benzimidazole thioether fragment.



Figure S5

1

H NMR spectrum of ESO

1

The H spectrum for the ESO starting material is shown with peaks labelled and integrated. Data were collected on a 400 MHz NMR in CDCl3 , corrected to TMS by residual non-deuterated solvent.

Figure S6

HMBC spectrum of ESO

The spectrum shows the long range coupling of 1 H to 13 C nuclei. Coupling is observed between the 3-methyl group and the AB system of ESO, and between the 5-methyl group and the pyridinyl methoxy and pyridinyl hydrogen.



Figure S7

1

H NMR spectrum of turnover products from ESO oxidation by the P450 BM3 F87V mutant

Only the product peaks are labelled and integrated for clarity. The spectrum shows the generation of a methoxy peak at δ 4.69 and a new methyl peak at δ 2.14, indicative of hydroxylation at one of the ESO methyl groups. The downfield shift of the pyridinyl hydrogen signal and the lack of shift in the AB system indicate hydroxylation at the 5 position. Data were collected on a 400 MHz NMR in CDCl3 , corrected to TMS by residual non-deuterated solvent.

Figure S8 HMBC spectra of turnover products from ESO oxidation by the P450 BM3 F87V mutant The data show the long range coupling of 1 H to 13 C nuclei. Additional peaks generated (by comparison with Figure S6 above) show coupling of the new methoxy peak (δ 4.69) to the pyridinyl methoxy (δ 3.67), the pyridinyl hydrogen (δ 8.37) and to the new methyl peak (δ 2.14). Lack of coupling to the AB system confirms that hydroxylation occurs on the 5-methyl (and not the 3-methyl) group.



Figure S9

1

H NMR spectrum of LAN

1

The H spectrum for the LAN starting material is shown with peaks labelled and integrated. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.

Figure S10

1

H NMR spectrum of the LAN sulfone standard

The 1 H NMR spectrum for the LAN sulfone standard is shown with peaks labelled and integrated. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.



Figure S11

1

H NMR spectrum of turnover product from LAN oxidation

Product was generated in the reaction of the F87V/A82F (DM) BM3 with LAN substrate. Only the product peaks are labelled and integrated for clarity. The spectrum shows the generation of a methoxy peak at δ 5.09 and a new methyl peak at δ 2.22, indicative of generation of the LAN sulfone. The aromatic region is largely unchanged, though with overlapping product and starting material peaks increasing the integrated values. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.

Figure S12

1

H NMR spectrum of RAB

1

The H spectrum for the RAB starting material is shown with peaks labelled and integrated. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.



Figure S13

COSY NMR spectrum of RAB

The COSY spectrum for the RAB starting material is shown. This was used to assign the methoxypropoxy carbon chain δ 1.98, 3.49 and 4.10. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.

Figure S14

HMBC spectrum of RAB

The spectrum shows the long range coupling of 1 H to 13 C nuclei. Coupling is observed between the 3-methyl group and the AB system, and between the various hydrogens on the alkyl chain.



Figure S15

1

H NMR spectrum of turnover products from RAB oxidation at 100 μM substrate

Products were generated in the reaction of the F87V/A82F (DM) BM3 with RAB substrate at 100 μM. The spectrum shows the total loss of the AB system to produce a singlet at δ 4.69. As this integrates for both protons, this indicates the loss of the sulfoxide oxygen to produce a thioether. The rest of the spectrum is unchanged, indicating that no significant further reaction took place. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.

Figure S16

1

H NMR spectrum of turnover products from RAB oxidation at 50 μM substrate

Products were generated in the reaction of the F87V/A82F (DM) BM3 with RAB substrate at 50 μM. The spectrum shows the total loss of the AB system to produce a singlet at δ 4.69. As this integrates for both protons, this indicates the loss of the sulfoxide oxygen to produce a thioether. There is also total loss of the methyl end of the methoxypropoxy group, indicating oxidative demethylation has taken place. The demethylation causes small shifts in the adjacent propyl chain signals, but otherwise the rest of the spectrum is unchanged, indicating that no significant further reaction took place. Data were collected on a 400 MHz NMR in DMSOd6, corrected to TMS by residual non-deuterated solvent.

Received 7 January 2014/3 March 2014; accepted 4 March 2014 Published as BJ Immediate Publication 4 March 2014, doi:10.1042/BJ20140030


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