Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1 Homology model guided site-directed mutagenesis Guillaume A. Schoch 1 , Roger Attias 2 , Monique Le Ret 1 and Danie ` le Werck-Reichhart 1 1 Department of Plant Stress Response, Institute of Plant Molecular Biology, Universite ´ Louis Pasteur, Strasbourg, France; 2 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite ´ Paris V, 45 Paris, France CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to iden- tify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydro- gen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivi- ties, supports the involvement of N302, I371 and K484 in substrate docking and orientation. Keywords: active site; cinnamate 4-hydroxylase; homology modeling; plant cytochrome P450; site-directed muta- genesis. CYP73 designates a family of plant cytochromes P450 that evolved with or before the evolution of vascular plants. Up to 20% of the woody plant biomass is processed by CYP73 enzymes to form lignin monomers, UV-shielding or insect attracting pigments, and defensive compounds [1,2]. CYP73 enzymes belong to the same subfamily, i.e. share more than 55% amino acid identity, and catalyse the regiospecific 4-hydroxylation of trans-cinnamic acid into p-coumaric acid [3–5]. The importance of this reaction in plant biology seems to have precluded further evolution and diversification of the CYP73A P450 subfamily to the processing of other endogenous metabolites.CYP73A1was one of the first plant P450 genes isolated [6]. Expression in yeast indicated that the cinnamate 4-hydroxylase (C4H) activity proceeds with a perfect coupling of oxygen consumption and reducing equivalents to produce hydroxylated substrates [3]. CYP73A1 provides a good model for determining the residues that control catalytic efficiency and optimal substrate positioning in a typical plant P450 enzyme contributing to a high throughput anabolic pathway. CYP73A1 is one of the most extensively studied plant P450 enzymes. It has a quite high substrate specificity but can accommodate a diverse array of compounds, as far as they are structural analogues of the natural substrate. Structural requirements for such analogues include a planar, aromatic structure, a small size of about two adjacent aromatic rings, and an anionic site opposite (i.e. at about 8.5 A ˚ ) to the position of oxidative attack [7,8]. A recent site- directed mutagenesis study that investigated the role of unusual residues in the most conserved regions involved in haem binding and oxygen activation [9], suggested that some are likely to contribute to the optimal coupling of the C4H reaction. The protein residues that govern substrate recognition and orientation have not yet been identified. In order to obtain information on the orientation and positioning of the substrates in the active site, we have recently engineered a stable and water-soluble form of CYP73A1 that is suitable for 1 H-NMR paramagnetic relaxation experiments [10]. The results of the NMR analysis indicated that the average initial orientation of the substrates in the catalytic site of the resting Fe(III) protein is roughly parallel to the haem. We decided to use a structure-based approach to site-directed mutagenesis in order to identify residues that affect substrate binding and turnover. However, only one structure for a membrane- bound P450 protein was available [11]. We thus had to rely on a homology model based on soluble P450 structures to predict residues that might participate in the substrate recognition and docking. In this paper, we report the Correspondence to D. Werck-Reichhart, Department of Plant Stress Response, Institute of Plant Molecular Biology, CNRS-UPR2357, Universite ´ Louis Pasteur, 28 rue Goethe, F-67000 Strasbourg, France. Fax: +33 3 90 24 18 84, Tel.: + 33 3 90 24 18 54, E-mail: daniele.werck@ibmp-ulp.u-strasbg.fr Abbreviations:CA,trans-cinnamic acid; C4H, cinnamate 4-hydroxy- lase; IAA, indole-3-acetic acid; I2C, indole-2-carboxylic acid; I3C, indole-3-carboxylic acid; 7MC, 7-methoxycoumarin; NA, 2-naphthoic acid; SRS, substrate recognition site. (Received 24 March 2003, revised 28 May 2003, accepted 2 July 2003) Eur. J. Biochem. 270, 3684–3695 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03739.x construction of a CYP73A1 model, the identification of residues likely to form contacts with the substrate, and the confirmation by site-directed mutagenesis of the involve- ment of some of these residues in the docking and catalysis of cinnamic acid. The impact of the active site mutations on the binding and metabolism of cinnamic acid analogues is also described. Experimental procedures Chemicals Trans-cinnamic acid (CA), trans-cinnamaldehyde, indole- 3-acetic acid (IAA), indole-2-carboxylic acid (I2C), indole- 3-carboxylic acid (I3C), 7-methoxycoumarin (7MC), 2-naphthoic acid (NA), phenylpyruvic acid, NADPH and umbelliferone were from Sigma-Aldrich (l’Isle d’Abeau Chesnes, France). trans-Cinnamylic alcohol and 6-hydroxy- 2-naphthoic acid were from Lancaster Synthesis (Stras- bourg, France). L (–)-Phenylalanine and naphthalene-1-acetic acid were from Merck (Schuchardt, Germany). 2-Amino- quinoline and 2-phenoxyacetamidine were from Maybridge (Tintagel, UK), 5-hydroxy-2-indolecarboxylic acid was from Acros Organics (Noisy-Le-Grand, France), trans- [3- 14 C]cinnamate was from Isotopchim (Ganagobie, France). 4-Propynyl-oxybenzoic acid was a gift from W. Alworth (Tulane University, New Orleans). Mutagenesis The modified CYP73A1 cDNAs were generated using QuickChange TM Site-Directed Mutagenesis (Stratagene) using as a template the double-stranded wild-type CYP73A1 cDNA from Helianthus tuberosus (GenBank Z17369) subcloned as an EcoRI–BamHI fragment into the shuttle vector pYeDP60 [12] and the primers listed in Table 1. PCR mixtures (40 lL) contained 250 l M of each dNTP, 0.5 l M each primer, 30 ng template DNA, 2.5 U Pfu DNA polymerase (Stratagene), 20 m M Tris/HCl pH 8.75, 10 m M KCl, 6 m M (NH 4 ) 2 SO 4 ,2m M MgSO 4 , 0.1% Triton X-100 and 10 lgÆmL )1 BSA. The polymerase was added after preheating for 2 min at 95 °C. Thirteen cycles of amplification (90 °C, 1 min; specific annealing temperatures for each set of primers given in Table 1, 90 s and 72 °C, 22 min) followed by 10 min extension at 72 °C. Parental methylated DNA was selectively digested with DpnI before transformation of Escherichia coli. The inserts of the selected neosynthetized vectors were fully sequenced. As neosynthetized DNA is not a template for the reaction, the amplification is linear, which is expected to keep the error frequency low in the final PCR product. Two problems were, however, encountered in our experiments: additional mutations around the site of mutagenesis and a large proportion of wild-type vectors were frequently obtained. As controls showed that the parental DNA was digested, this was attributed to poor primer synthesis or correcting properties of the polymerase. Yeast expression and microsome preparation The pYeDP60 vector [12] and the modified strain of Saccharomyces cerevisae W(R) over-expressing its own NADPH-P450 reductase were used for the expression of the constructs [13]. Yeast transformation was performed as described in [14], growth and induction were based on the high density procedure described in [15]. To achieve optimal expression, a yeast colony grown on an SGI plate was tooth-picked into 50 mL SGI and grown for 18 h at 30 °C to a density of 6 · 10 7 cellsÆmL )1 . This preculture was diluted in YPGE to a density of 2 · 10 5 cellsÆmL )1 ,and grown for 30–31 h until it reached a density of 8 · 10 7 cellsÆmL )1 . Protein expression was induced by addition of 10% aqueous solution of galactose at 200 gÆL )1 .Final density after 17 h of induction at 28 °C was routinely around 2 · 10 8 cellsÆmL )1 . Microsomal membranes were isolated by ultracentrifugation after mechanical disruption of the yeast cells with glass beads [15]. Microsomes from W(R)transformedwithvoidpYeDP60wereusedasa negative control. Spectrophotometric measurements and catalytic activity P450 content was calculated from CO-reduced vs. reduced difference spectra [16]. Low to high-spin conversion and Table 1. PCR primers used for site-directed mutagenesis. The DKR primer, meant to generate K248T/R249M double mutants, actually produced the D247E/K248T/R249M and K248T/R249M/I371K triple mutants. Mutant Sense Primer Antisense Primer T m R101M 5¢-GAGTTTGGTTCGATGACAAGGAATGTTG-3¢ 5¢-CAACATTCCTTGTCATCGAACCAAACTC-3¢ 58 R103M 5¢-GTTCGAGAACAATGAATGTTGTGTTC-3¢ 5¢-GAACACAACATTCATTGTTCTCGAAC-3¢ 55 R103E 5¢-GAACACAACATTCTCTGTTCTCGAACC-3¢ 5¢-GGTTCGAGAACAGAGAATGTTGTGTTC-3¢ 55 DKR 5¢-GAAGTTAAAGATACAATGATTCAGCTC 5¢-GAGCTGAATCATTGTAACTTTAACTTC-3¢ 48 N302D 5¢-CATTGTTGAAGACATCAATGTTG-3¢ 5¢-CAACATTGATGTCTTCAACAATG-3¢ 43 N302F 5¢-CTTTACATTGTTGAATTCATCAATGTTGCAGC-3¢ 5¢-GCTGCAACATTGATGAATTCAACAATGTAAAG-3¢ 43 I303A 5¢-CATTGTTGAAAACGCTAATGTTGCAG-3¢ 5¢-CTGCAACATTAGCGTTTTCAACAATG-3¢ 52 R366M 5¢-CAAGGAAACCCTCATGCTCCGTATG-3¢ 5¢-CATACGGAGCATGAGGGTTTCCTTG-3¢ 55 R368K 5¢-CCCTCCGTCTCGAAATGGCGATCCG-3¢ 5¢-CGGGATCGCCATTTCGAGACGGAGGG-3¢ 50 R368F 5¢-CCCTCCGTCTCTTTATGGCGATCCG-3¢ 5¢-CGGGATCGCCATAAAGAGACGGAGGG-3¢ 50 I371F 5¢-TCCGTATGGCGTTCCCGCTTCTAGTC-3¢ 5¢-GACTAGAAGCGGGAACGCCATACGGA-3¢ 58 I371A 5¢-TCCGTATGGCGGCTCCGCTTCTAGTC-3¢ 5¢-GACTAGAAGCGGAGCCGCCATACGGA-3¢ 58 I371K 5¢-TCCGTATGGCGAAACCGCTTCTAGTC-3¢ 5¢-GACTAGAAGCGGTTTCGCCATACGGA-3¢ 58 K484M 5¢-GATACCGATGAGATGGGTGGGCAGTTTAG-3¢ 5¢-CTAAACTGCCCACCCATCTCATCGGTATC-3¢ 58 Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3685 dissociation constants of enzyme–ligand complexes were evaluated from Type I ligand binding spectra using the e peak-trough ¼ 125Æm M )1 Æcm )1 [7]. Integrity of the enzyme was checked at the end of each titration experiment by recording a difference spectrum of the CO-reduced protein. Cytochrome c reductase activity of the NADPH-cyto- chrome P450 reductase was assayed as in [17]. Trans-CA hydroxylation was assayed using radiolabelled trans- [3- 14 C]CA and TLC analysis of the metabolites [18]. For determination of the kinetic constants, data were fitted using the nonlinear regression program DNRPEASY derived from DNRP53 [19]. I2C and I3C hydroxylations were assayed in a total volume of 200 lL 100 m M sodium phosphate pH 7.4 containing 600 l M NADPH, 100 l M substrate and 70 lg yeast microsomal protein. Incubations, at 27 °Cfor20min for measurement of catalytic activity and for 90 min for products identification, were stopped by the addition of 20 lL 4 N HCl. Reaction products were extracted three times with two vols ethyl acetate, the organic phases were pooled and evaporated under argon. The residue was dissolved in acetonitrile, water, acetic acid (10 : 90 : 0.2, v/v/v) and analysed by reverse-phase HPLC (LiChrosorb RP-18 Merck, 4 · 125 mm, 5 lm); flow rate 1 mLÆmin )1 ; 5 min isocratic, then 20 min linear gradient from 10 to 52% acetonitrile. Negative controls incubated with W(R) yeast microsomes were used to test for CYP73-independent reactions and to evaluate extraction yields. Product elution was monitored by photodiode array detection. Retention times of I3C and its oxygenated product were 13 and 6.5 min, respectively. Products of I2C incubation were collected, evaporated and submitted to MS analysis on a BioQ triple quadrupole (Micromass). Phenylalanine, which is insoluble at pH 7.4, was dissolved in sodium borate 100 m M pH 8.3. Phenylalanine and 2-phenoxyacetamidine hydroxylations were assayed by HPLC under similar conditions as I2C and I3C, excepted for phenylalanine mobile phase (isocratic 5% acetonitrile, 7.5 m M (NH 4 ) 2 PO 4 ,7.5m M HCl). NA hydroxylation was assayed by fluorometry [7] in 2 mL 100 m M sodium phosphate pH 7.4 containing 0.2, 0.5 or 1 mg yeast micro- somal protein, 600 l M NADPH, and 100 l M substrate. Product formation was monitored for 10 min at 30 °C. 7MC hydroxylation was assayed as in Werck-Reichhart et al. [20] with 1 mg microsomal protein in the assay. Modelling programs and calculations Calculations were carried out on an Indy Silicon Graphics computer. Common structural blocks were determined previously using the GOK interactive program [21]. Side chain atoms determination, distance and dihedral con- straints calculation, rotamer selection, and data analysis were performed by writing macros in BCL language from Accelrys (MSI), in AWK language, in UNIX macros, and by using the functionalities of INSIGHTII and BIOPOLYMER modules from Accelrys. The program DYANA that calculates the initial minimized model, was designed for NMR applications. It was modified (mainly the array sizes) in order to handle the large number of constraints generated by this method (about 35 000 constraints were kept for the present application). The input data to the modified DYANA program are then no longer NMR constraints, but geometrical distances and torsions derived statistically from the templates. DYANA minimization includes Van de Waals’ interaction calculations, and proposes its best solution from a starting conformation. Structures were analysed by using the PROCHECK package and Accelrys INSIGHTII . Model minimization was further refined with the functionalities of Accelrys DISCOVER 3 (version 97.0, Force Field CVFF and ESFF when including the haem iron atom). At this stage, electrostatic interactions are included in the minimization process. At each of the modelling steps, models are selected on the basis of quality scores supplied by the related program (f factor in DYANA , or PROCHECK G-factors scoring ideally above )0.5 for instance). Construction of the models Homology models of cytochrome P450 CYP73A1 were constructed using building blocks corresponding to com- mon P450 three-dimensional substructures (or common structural blocks) of the four structures (P450 BM3 , P450 CAM ,P450 TERP ,andP450 eryF ) available from the Brookhaven PDB at the start of this work as entries 2HPD, 3CPP, 1CPT and 1OXA, respectively. Common structural blocks were determined for the four structures by Jean et al. [21] using the program GOK and the related strategy. For specified tolerance parameters, this program performs a multiple structure comparison from internal coordinates (we used Alpha, Tau). Consensus sequence of the blocks were then independently located in CYP73A1 using a multiple alignment of the available CYP73 sequences. For assigning three-dimensional coordinates to the common structural blocks in CYP73A1, we used a procedure implemented for modelling the CYP2Cs [22], and based on the adaptation of a technique designed for deriving structures from NMR data [23]. The atoms of the side chains showing identical spatial location when superimposing each set of residues were considered as conserved atoms. They were identified and added to the list of the block backbone conserved atoms. These side chain atoms also provide the resulting rotamer value for the related target residue. Other rotamers, for residues with no conserved side chain atom, were attributed by using a rotamer library [24]. From the three-dimensional coordinates of the common structural blocks, we derived a set of geometrical constraints (mean distances between two atoms, mean Phi and Psi values), and their standard deviations. The distance cutoff between two atoms was set to 5 A ˚ , except for interblock CB atoms where no cutoff was given in order to reflect the more flexible relative location of the blocks. These constraints constitute, within a tolerance interval, the spatial informa- tion that was used to build the model. The DYANA program was then used to calculate initial random coordinates of the target protein and performed minimization under this set of distance and dihedral constraints [25]. The loops between the blocks were built with no constraints. From each model, Phi and Psi additional constraints for nonconserved residues were derived in order to restrain them in an allowed region of the Ramachandran region. DYANA was then rerun and proposed a family of models. Minimization refinements and 3686 G. A. Schoch et al. (Eur. J. Biochem. 270) Ó FEBS 2003 docking were performed for a set of selected models. The PROCHECK program [26] was applied as a help to the selection of the models. Results Modelling the active site of CYP73A1 CYP73A1 does not show strong identity with any of the P450 proteins that have been crystallized. Also, it does not reliably align with the sequences of known structures in areas other than the most conserved regions common to all P450 enzymes. A CYP73A1 homology model was built using the computational strategy, previously described by Jean et al. [21] and further improved by Minoletti [22], that identifies substructures, or structurally conserved blocks, in the crystal structures of related proteins, and then locates similar blocks in the target sequence. Common structural blocks of the four P450 structures (P450 BM3 ,P450 CAM , P450 TERP , and P450 eryF ) available from the Brookhaven PDB at the start of this work were located in the CYP73A1 sequence as represented in Fig. 1. Common structural blocks were used to assign three-dimensional coordinates to the corresponding blocks in CYP73A1. The resulting model of the CYP73A1 core structure and active site region is represented in Fig. 2. The advantage of this approach is that it merges structural information from several known structures into the target protein rather than producing a model that is based on a single structure. All techniques are limited by the prediction of the protein alignments, but integration of information from multiple structures has some chances to be better when, as in our case, protein identities are very low. The 6–8 A ˚ distances between the substrate protons and the haem iron were recently deduced from 1 H paramagnetic relaxation experiments [10] indicate that CA initially binds roughly parallel to the haem in the oxidized CYP73A1. The carboxylic function, which can be replaced by other anionic groups, was previously shown to be an essential determinant of substrate docking in the active site [7,8]. An ionic or hydrogen bond is likely to anchor CA to a cationic or hydrophilic residue of the protein. These data suggest that a set of residues within 5–9 A ˚ above haem iron could be considered putative active site contacts and tested by site- directed mutagenesis. A search of the model for hydrophilic residues likely to form a hydrogen bond with the substrate pointed to N302 in the I helix as a good carboxylate binding candidate as it is one turn away from the so-called oxygen groove. A set of cationic residues that were predicted to reside in the substrate-binding regions (substrate recognition sites or SRS [27]), in particular SRS 1, 3 and 5, on the basis of a multiple alignments with bacterial and mammalian enzymes, were also chosen for mutagenesis to circumvent model-prediction inaccuracy. Based on SRS predictions, the modified cationic residues included R101, R103, K248, R249, R366, R368 and K484. Only K484 was predicted in the substrate pocket in our model. However, its distance to the haem seemed too large to allow direct interaction with the substrate anionic site. Hydrophobic contacts with the aromatic ring of the substrate were also investigated. A306 modification was previously shown to adversely affect the binding of cinna- mate and the coupling of the hydroxylation reaction [9]. This effect was probably due to a direct interaction of its side-chain with the aromatic ring of the substrate. Our model supports this hypothesis. The model predicts that I371 is another residue in close proximity to the substrate. I371 aligns with F361 in the limonene 6-hydroxylase, a residue that was shown to control the regioselectivity of limonene hydroxylation by CYP71Ds [28]. Finally, I303 is located close enough to the putative substrate pocket to form a hydrophobic contact. However, such a contact would be precluded in the hypothesis of a van der Waals’ interaction with I371 and a hydrogen bond to N302. Substitute residues were chosen to alter charge and hydrophilicity with minimal change alteration to side chain Fig. 1. Predicted location of the conserved structural blocks and SRSs on the primary sequence of CYP73A1. Sequence alignments of CYP73A1 with the common structural blocks of four bacterial crystal structures (P450 BM3 , P450 CAM , P450 TERP ,andP450 eryF )predicted some of the substrate recognition sites regions. SRS locations were corroborated on the basis of a multiple alignment with the four bac- terial enzymes also including some members of the CYP2 and CYP73 families. CYP73A1 putative SRSs determined on the basis of this alignment are underlined (numbered 1–6 from N to C terminal) and residues selected for directed mutagenesis are indicated by stars. The region interblocks in CYP73A1 are displayed in grey. For the bacterial sequences only the common structural blocks are represented, the identity between sequences is shaded in black, similarity is shaded in grey (threshold of 70%). Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3687 bulk, except in the case of hydrophobic contacts for which the influence of side-chain size was investigated. The consecutive residues K248 and R249 were modified simul- taneously to avoid charge compensation. As the desired double mutations were not obtained, we analysed the triple mutants D247E/K248T/R249M (DKR) and K248T/ R249M/I371K (KRI). Impact of mutations on the structure and stability of the protein The impact of amino acid substitutions on protein stability was investigated using the initial CO-difference spectra of the reduced enzyme to quantify amounts of properly folded protein with correct incorporation of haem. The time- and temperature-dependent disappearance of the peak at 450 nm was monitored as well as any conversion of P450 into P420 that would reflect disruption of the haem–thiolate bond [29], to test the stability of the core structure. High and fast P450 disappearance usually correlated with decreased yeast expression and indicated a link between improper folding or stability loss and expression levels of the mutant protein. Immunoblot quantification of the apoprotein content in yeast microsomes using antibodies raised against purified CYP73A1 [30] revealed decreases in polypeptide expression of the mutants that did not exceed 40% compared to the wild-type construct. Carbon monoxide difference spectra detected the presence of haem in all of the mutants, although very low CO-binding was obtained with R366M, R101M or for the triple mutants (Table 2). The modifica- tions to residues I303, I371, R103 and K484 did not appear to affect the production of haem protein. P450 disappearance followed pseudo-first order kinetics. Under standard conditions, i.e. when P450 spectra were recorded in the presence of 0.5 mgÆmL )1 sodium dithionite and 30% glycerol, the half-life (t 1/2 ) of the wild-type CYP73A1 was around 3 h. In the presence of a higher sodium dithionite concentration (4.5 mgÆmL )1 ), the t 1/2 of CYP73A1 was 45 min when the buffer contained 3% glycerol, and 60 min with 30% glycerol. Stability tests were performed at 4.5 mgÆmL )1 dithionite, using different con- centrations of glycerol depending on the stability of each mutant (Table 2). The results identified three classes of mutants. The first group consisted of N302D, I371F and I371K, that had a stability at least equal to that of the wild- type. The second group included K484M, with a stability that was slightly decreased compared to the wild-type, and R103M, N302F and I371A that displayed a more pro- nounced decrease with a t 1/2 shift from 45 to approximately 15 min. The third group, included all other mutants in particular R366M and R101M, which demonstrated a drastic loss in protein stability. The R366M, R368F/K, R101M, R103M/E, DRK and KRI modifications resulted in a very significant disruption of the tertiary structure of the CYP73A1 protein. Effect of mutations on cinnamic acid recognition and metabolism The impact of the mutations on CA binding and metabo- lism was investigated (Table 2). The K484M mutation that Fig. 2. A preliminary model of the active site of CYP73A1. Construction of this first model was based on four bacterial crystallized structures. Only part of the active site is shown. Based on the 1 H-NMR data [10], the substrate is expected to be located between the spheres. Generated by using SWISS - PDB viewer andrenderedwith POV - RAY . 3688 G. A. Schoch et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Table 2. Impact of the mutations on protein stability and CA recognition and metabolism. Expression levels were calculated from CO-difference spectra. The initial proportion of P420 was estimated from these spectra. Stability of the haem protein was assayed by monitoring the disappearance of the 450 nm peak from the CO difference spectra in recombinant yeast microsomes reduced with high sodium dithionite (4.5 mgÆmL )1 ). The half-lives (t 1/2 ) calculated from the pseudo-first order kinetics of P450 decrease are reported. Spectra were recorded every 0.5 or 1 min during 30 min. (1) Microsomes were incubated at 30 °C in sodium phosphate 100 m M pH 7.4 containing 3% glycerol. (2) Low stability mutants were tested in buffer containing 30% glycerol to underline the differences between them. C4H activity was measured using a concentration of cinnamate (150 l M ) expected to be saturating for most of the mutants. The binding constants were calculated from the amplitude of the type I difference spectra induced by increasing concentrations of substrate, e type I being the molar absorption coefficient of the saturated P450-substrate complex (DA max /P450 concentration) and K s the dissociation constant. Expression and activity values are relative to the wild-type (100%): P450 expression, 847 pmolÆmg )1 microsomal protein; C4H activity, 287 pkatÆmg )1 ; cytochrome c reductase activity, 1520 pkatÆmg )1 . Cytochrome c reductase activity is used as a control for protein induced expression and integrity. Values ± SD are the mean of three or more experiments. n.m. not measurable. Hydrophobic and hydrogen bonding residues Positively charged residues I helix (SRS 4) Loop 3 (SRS 5) B helix (SRS 1) (SRS 3) K helix (SRS 5) (SRS 6) Wild-type N302F N302D I303A I371F I371A I371K Wild-type R101M R103M R103E DKR KRI R366M R368K R368F K484M Yeast expression level (%) 100 ± 4.8 30 ± 0.3 71 ± 7.2 96 ± 5.1 95 ± 9.9 93 ± 2.8 105 ± 2.4 100 ± 4.8 11.2 ± 0.7 60 ± 3.5 78 ± 1.7 7.8 ± 0.2 11.6 ± 0.8 <5 81 ± 3.3 54 ± 4.3 89 ± 1.4 Initial P420 (%) – 15 – <5 – – – – 50 – 5 65 <5 >80 – – – t 1/2 (min) (1) 46 ± 7.5 13.8 ± 3.9 45.1 ± 5.7 5 ± 1 53.2 ± 7.1 12.7 ± 0.8 53.9 ± 7.6 46 ± 7.5  2 15 ± 4.7  2<1 2 n.m.  2  2 30.7 ± 0.6 t 1/2 (min) (2) 59.8 ± 7 10.2 ± 4.8 – 6.7 ± 0.7 3 ± 2 8.9 ± 0.9 n.m. 26± 2.4 13 ± 1.2 – C4H activity (%) 100 ± 1.0 0.5 ± 0.2 10 ± 0.9 75 ± 4.6 0.09 ± 0.02 11.3 ± 1.5 1.1 ± 0.2 100 ± 1.0 0.2 ± 0.3 44 ± 4.5 36 ± 5.8 1.0 ± 0.1 0.1 ± 0.05 0.1 ± 0.1 60 ± 3.5 48 ± 4.1 55 ± 6.1 Cinnamate binding K s (l M ) 7.1 ± 1.0 13.7 ± 2.6 45 ± 9.0 3.9 ± 0.3 >100 25 ± 3.0 11.1 ± 2.2 7.1 ± 1.0 no type I 16.7 ± 2.1 >50 5.2 ± 1.6 11.9 ± 0.7 >100 11 ± 0.5 11 ± 0.6 5.9 ± 0.2 e type I (m M )1 Æcm )1 ) 128 ± 7.5 23 ± 4.7 7.5 ± 1.0 106 ± 3.0 1.0 ± 0.5 25 ± 1.5 15.6 ± 2.3 128 ± 7.5 – 120 ± 9.9 103± 2.2 35 ± 3.8 23 ± 9.2 n.m. 133 ± 6.6 126 ± 5.7 112 ± 0.9 Cyt c reductase activity (%) 100 ± 5.0 98 ± 6.9 166 ± 5.9 107 ± 4.9 101 ± 7.2 146 ± 13 106 ± 12 100 ± 5.0 91.1 ± 21 136 ± 14 119 ± 17 83 ± 1.7 100 ± 13 118 ± 6.0 102 ± 7.3 98 ± 6.4 125 ± 9.8 Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3689 did not significantly affect protein expression or stability had no significant impact on the binding of CA; however, it did result in a 45% decrease in catalytic activity. All other modifications of positively charged amino acids adversely affected expression and/or stability of the enzyme but had a comparatively minor affect on substrate recognition and metabolism. Exceptions included R366M, R101M and the triple mutations for which drastic decreases in stable haem protein were paralleled by dramatic losses in activity. Despite the loss of activity and structural integrity, the DKR mutation rather unexpectedly seemed to retain an intact affinity for substrate binding. Modifications of N302 and I371 resulted in limited or no apparent perturbation of protein folding and stability but led to dramatic decreases in CA binding and hydroxylation. N302 is likely to provide a hydrogen bonding side chain for anchoring the carboxylate of CA. The conversion of aspa- ragine into negatively charged aspartic acid (N302D) resulted in a drastic effect on substrate binding affinity. Whereas replacement with a bulky hydrophobic residue (N302F) compromised overall protein structure and cata- lysis. I371 is predicted to form a van der Waals’ contact with the aromatic ring of CA. In the I371 mutants, I371A opens more space in the active site and thus should allow for increased substrate mobility. Conversely, I371F and I371K should create a steric hindrance to the binding of the substrate above the haem iron. As expected, the I371A mutation substantially decreases CA affinity and the ability to desolvate the active site. Around 10% of the catalytic activity is conserved, which would be in agreement with the conservation of the carboxylate anchoring function of the protein. The I371F and I371K mutations lead to an almost complete loss in C4H activity. This activity loss is correlated with impaired substrate binding. A complete loss of binding was also observed upon substitution of I371 with the bulky phenylalanine. The insertion of a positive charge in the 371 position does not completely prevent the binding of the substrate, but almost totally hinders catalysis. This probably results from improper positioning of the substrate’s aro- matic ring above the haem iron. Mutation of I303, adjacent to N302, into alanine slightly increased affinity but modified substrate positioning and decreased catalytic activity. This data is concordant with a model where I303 is not a direct contact residue, but rather contributes to optimal CA orientation in the active site. Binding of alternate ligands to CYP73A1 mutants The mutants that showed strongly impaired CA binding and metabolism, but that did not display a major structural alteration in terms of protein stability and expression, were further tested for their ability to recognize a set of structural analogues of CA. This set included CA precursors, plus other natural and synthetic compounds. Some of these compounds present a quite high intrinsic affinity for wild- type CYP73A1, such as phenylpyruvic acid (K s ¼ 3.1 l M ), phenylalanine, indole-2-carboxylic acid or cinnamyl alcohol (K s ¼ 12 l M ), 2-aminoquinoline (K s ¼ 17 l M ) and indole- 3-carboxylic acid (the natural auxin, K s ¼ 18 l M ). These compounds are ordered from gain to loss of binding to the mutant proteins in Table 3. As shown in Table 3, the analogues investigated were better ligands for the mutants than the physiological substrate CA. Relative to wild-type CYP73A1, the binding efficiency for CA decreases 10-fold in the mutant I371K, 50-fold in I371F and 100-fold in N302D. In contrast, increases in binding efficiency are observed for a few ligands after modification of the protein. The most notable increases are 15-fold for N302D with phenylalanine, 12-fold for I371K with 2-phenoxyacetamidine, and 10-fold for I371F with phenylalanine or cinnamylic alcohol. The I371F modification is likely to block access to the active centre for most of the potential substrates. Only compounds with increased side chain flexibility or reduced bulkiness in the CA ring region are expected to have increased binding efficiencies compared to CA. This is actually the case, with a gain in binding efficiency being observed only for phenylalanine, 2-phenoxyacetamidine, cinnamylic alcohol or 4-propynyl-oxybenzoic acid. More relevant are the N320D and I371K mutations that could provide a new salt-bridge or hydrogen bonding opportu- nities in the active site region. Increased affinity of several ligands indicates that new bonds are formed in the mutants and may reflect a reversed orientation of the ligands or occupation of different subpockets in the active site. Noteworthy are the increased binding of phenylalanine and 2-phenoxyacetamidine, which are highly polar mole- cules. However, none of the compounds that displayed an increased affinity produced a large spin transition of the ferrous haem, which would be indicative of effective desolvation of the active site and appropriate positioning for an efficient oxidative attack. Some of the analogues listed at the top of Table 3 that showing better binding efficiencies than CA with the modified proteins, were analysed in catalytic assays. Metabolism of alternate substrates The metabolism of CA analogues was assayed with the N302, I371 and K484 mutants (Table 4). Microsomes from yeast transformed with the empty expression plasmid, and also incubations without NADPH were used to control for CYP73-independent reactions. No metabolism of phenyl- alanine and 2-phenoxyacetamidine was detected with the wild-type or any of the mutants. The sensitivity of the tests was low, due to high detection thresholds and the need to test phenylalanine metabolism at pH 8.3, which decreases C4H activity of the wild-type by 80%. NA was previously shown to be the best structural mimic and alternate substrate for wild-type CYP73A1 [7]. NA was metabolized by all mutants with an efficiency very compar- able to that observed with CA. This suggests that both compounds have a very similar positioning in the active site and validates use of NA for fluorometric quantification of the enzyme activity [7]. Metabolism of I2C, I3C and 7MC does not parallel that of CA in the different mutants. For example the I371A and I371K mutations have less influence on demethylation of 7MC than on CA hydroxylation. Also noteworthy is the opposite effect of several amino acid substitutions on I3C and I2C hydroxylations. Most muta- tions have less impact on I2C than on I3C and CA metabolism, probably due to the symmetry axis of I2C and to the possible attack on two different carbon atoms. 3690 G. A. Schoch et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Unexpectedly, the K484M substitution, which results in close to 50% loss in C4H activity, does not affect I2C hydroxylation. As initial cinnamate binding is not influ- enced by this mutation (Table 2) and binding kinetics are first-order (indicating a single binding-site), this suggests that K484 does not directly affect catalysis but might have a selective role in substrate position adjustment during the catalytic cycle. Modified regiospecificity of indole-2-carboxylic acid hydroxylation I2C metabolism by wild-type CYP73A1 was previously shown to result in the formation of two products that were not further characterized [7]. On the basis of its HPLC retention time, UV spectrum, and monoisotopic mass, the most polar product P1 (RT 9.8 min) was unambiguously identified as 5-hydroxy-I2C (Fig. 3). P2 presents the same mass as P1 and is thus a monohydroxylated product. Superimposition of the NA and CA structures, and of their positions of attack on those of I2C, indicates that P2 is most likely 6-hydroxy-I2C, although an authentic standard was not commercially available for verification. In favour of the latter hypothesis, 5-hydroxy-I2C was tested as a substrate of CYP73A1 and was not further metabolized. As preliminary experiments indicated that the ratio between the two products varies upon metabolism by the different mutants, this ratio was used as a reporter of the influence of the mutations on substrate docking (Table 4). In the wild-type CYP73A1, the formation of P2 is five times more frequent than that of P1. The K484M mutation does not significantly affect the P2/P1 ratio. This is not surprising considering that it does not affect the global rate of I2C metabolism. As the length of the I2C molecule and the distance between its carboxylate and the positions of attack are slightly shorter than for CA or NA, it is possible that the carboxylate of I2C is beyond the area of influence of K484. The N302 mutations, in particular N302D, significantly increased the proportion of P1 so that the P2/P1 ratio dropped closer to 1. This loss in regiospecificity in the mutant is concordant with the increased mobility of the Table 3. Alternate ligand binding to the mutant protein. 4-Propynyl-oxybenzoic acid and wild-type CYP73A1 is the only complex for which data fitting with the Michaelis–Menten equation indicated second order kinetics. Binding efficiency is the e type I /K s ratio calculated for each complex. The values listed are relative to the wild-type for each ligand. Standard deviations (not shown) are less than 12% of these values. Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3691 molecule in the active site that is reflected by a low e type I (Table 3). Taken together, the data support a role for N302 in controlling of substrate orientation in the active site. The I371 mutations to K and A have opposite effects. The I371K mutation increases the preferential attack at the putative 6-position, most likely by increasing steric hin- drance near C5 of the indole ring. In contrast, the I371A mutation appears to remove the steric constraint existing in the wild-type CYP73A1 and favours a P2/P1 ratio closer to 1. The observed effects of both of these mutations support the assumption of a direct contact of I371 with the aromatic ring of I2C or CA. Discussion The computational homology modelling strategy des- cribed by Jean et al. [21] allows a reasonable prediction of the most conserved P450 substructures, although hypervariable regions cannot be predicted. Our present model was based on four crystallized bacterial enzymes (Fig. 2) and seems to correctly predict several residues forming contacts with CA. The model predicts that N302, which resides in the I helix and SRS 4, is likely to form a hydrogen bond with the carboxylate of the substrate. Mutations of this residue lead to a dramatic loss in CA binding efficiency (10-fold for the N302F and 100-fold for the N302D substitution) together with a very strong decrease in catalytic activity. This confirms a critical role for this residue in the initial binding and correct positioning of CA during catalysis. A role of N302 in anchoring the side-chain carboxylate of CA is further supported by the enhanced binding of amine substituted ligands and also by the loss of regiospecificity of I2C hydroxylation when N302 is replaced by an aspartic acid. Together with A306, I371 is predicted to form a hydrophobic pocket that positions the aromatic ring of the substrate in close proximity to the haem iron. The adverse impact of the A306G substitution on substrate binding and metabolism as well as coupling of the reaction was described previously [9]. Modifications of I371, espe- cially I371F, produced a dramatic loss in binding and activity with CA and all other substrates. The less detrimental effect of these substitutions on the binding of analogues, which are less rigid or bulky than CA, and differential impact on the regiospecificity of I2C ring- hydroxylation support the hypothesis that the side chain of I371 is an essential element ensuring correct positioning and orientation of the aromatic ring in the active site. Our model predicts that K484 is in the substrate pocket. Its distance to the CA carboxylate in the oxidized enzyme model does not allow for any direct interaction and, as expected, modification of K484 has no impact on the initial binding of CA. However, the K484M substitution leads to a 50% decrease in catalytic activity with both CA and NA. A possible explanation is that K484 plays some role in the electron transfer from the P450 reductase to the haem iron. However, this residue is located on the distal side of the haem, while interaction with the reductase and electron transfer should involve residues on the proximal side of the protein [31]. The unchanged I2C hydroxylase activity in K484M when compared to that of the wild-type confirms that the mutant is not impaired in electron transfer. Thus, K484 must exert some control on CA/NA positioning or product release during the catalytic cycle. Although the K484 effect might be indirect and the interaction with the carboxylate of CA might occur via a molecule of solvent, it can also be postulated that the reduction of the protein or binding of oxygen results in a conformational change of the Fig. 3. Analysis of the products of I2C hydroxylation. Upper panel: HPLC analysis of the products of the metabolism of 10 nmol I2C by 30 pmol recombinant CYP73A1 in 60 min and in a 100 lL assay. Absorbance was monitored at 290 nm. Lower panel: UV spectra corresponding to the centre of the peaks. P1 and P2 collected after 90 min incubation of 120 nmol of I2C were analysed by negative ESI- MS. Monoisotopic mass of both compounds was 176 Da. P1 retention time and UV spectrum was identical to that of commercial 5-hydroxy- 2-indolecarboxylic acid. Table 4. Metabolism of alternate substrates by mutant CYP73A1s. Activities are expressed relative to wild-type CYP73A1. 100% activity is 287 pkatÆmg )1 microsomal protein for CA, 311 pkatÆmg )1 for NA, 38.8 pkat mg )1 for I2C, 20.4 pkat mg )1 for I3C, 6.6 pkat mg )1 for 7MC. n.d. not determined. Mutant CA NA I2C P1/P2 I3C 7MC 73A1 % 100 % 100 ± 2.3 % 100 ± 1.1 (5.7) % 100 ± 2.0 % 100 ± 8.2 N302D 10 9.7 ± 7.0 14.6 ± 0.8 (1.5) 3.0 ± 0.4 8 ± 1.5 N302F 0.5 2.3 ± 1.2 £ 1 (2.3) n.d. n.d. I371F 0.09 < 0.6 < 0.1 n.d. n.d. n.d. I371A 11 12 ± 0.8 32.1 ± 2.2 (0.7) 9.3 ± 0.3 27 ± 3.4 I371K 1.1 < 0.6 5.9 ± 0.5 (8.0) £ 1 6 ± 0.9 K484M 55 50 ± 3.8 94.3 ± 3.8 (4.9) n.d. n.d. 3692 G. A. Schoch et al. (Eur. J. Biochem. 270) Ó FEBS 2003 protein, similar to that observed for P450 BM3 or P450 CAM substrate complexes [32–34]. Such a change could bring K484 much closer to CA. In this case, ion pairing or hydrogen bond between K484 and the CA carboxylate could control the optimal positioning and orientation of the substrate for catalysis. NMR measurement of the distances of the substrate protons to the haem iron indicate an initial positioning of CA approximately 6–8 A ˚ from the iron in the oxidized enzyme, which might not be optimal for catalysis and would not particularly favour ring 4-hydroxylation. If these measurements are correct then a structural change that brings CA closer to the iron and adjusts substrate position, possibly tilting the substrate so as to favour attack at the 4 position or on the 3–4 bond, would be needed for efficient and regiospecific catalysis. The K484M mutation has no impact on I2C metabolism or the regioselectivity of attack. This observation is compatible with a role of K484 in CA reorientation as the slightly smaller size and different shape of I2C compared to that of CA might prevent interaction between its anionic site and K484. N302 and I371 align with residues that have been shown to confer substrate specificity or regioselectivity to many other plant or mammalian P450 enzymes. Residues corresponding to I371 govern the regiospecificity of the hydroxylation of 4S-limonene in CYP71D18 from spear- mint and CYP71D15 from peppermint for the synthesis of carvone and menthol, respectively [28]. In the mammalian CYP2B family, residues 294 and 363 are equivalent as N302 and I371, respectively. The CYP2B mutations were shown to affect steroid regioselectivity. At position 363, a CYP2B1 mutant (V363L) exhibited a twofold decrease in androgen activity [35], whereas in CYP2B11 the reverse mutant shows a fivefold increase in androgen activity [36]. The same residue was identified as a determinant of substrate specificity in CYP2B2 [37], CYP2B5 [38] and CYP2B6 [39]. Likewise, residue 294 was shown to play a key role in androgen metabolism by CYP2B1 [40] and CYP2B4 [38]. A similar affect on catalysis by these residue positions has been reported for other mammalian enzymes. For example in CYP2A5, mutation of M365, the equivalent of I371, decreased the metabolism of aflatoxin B1 [41], while modification of the corresponding residue (A370) in human CYP3A4 enhanced the hydroxylation of steroids [42,43]. A significant portion of the protein, which was not reliably predicted in the model, is not shown in Fig. 2 and was not thoroughly investigated in our site-directed experi- ments. It is therefore likely that additional residues, such as R or K that can form an ion pair with the carboxylate of CA, may contribute to substrate recognition or docking. Mutation of positively charged residues found in the putative SRSs (Fig. 1), based on a multiple alignment did not lead to the identification of a residue that would be critical for the recognition or positioning of CA. Mutation of all arginines led to a significant loss in protein stability suggesting that they are involved in protein fold structure rather than binding of the substrate. The overall picture of the CYP73A1 active site provided by our data is reminiscent of P450 BM3 [44,45], as it involves a hydrogen bond and possibly an ion pair for the anchoring of the carboxylate on the substrate, and also a major hydrophobic region for the docking of the aromatic ring. As in P450 BM3 , a substantial protein rearrangement must occur during the catalytic cycle [32,33], probably upon reduction, to ensure an optimal positioning of the substrate relative to the ferryl-oxo intermediate for coupled, regiospecific attack of the ring at the 4 position. While mutant analysis was in progress, the first X-ray structure was described for a membrane-bound mammalian P450, CYP2C5 [11]. This new structure confirmed the conservation of the P450 spatial organization in eukaryotic microsomal enzymes. The position of SRS 4 that is located in the centre of the I helix, which includes N302 in CYP73A1, was highly conserved relative to the haem. However, significant local changes were detected, particularly in all other SRSs. For example, SRS 5, facing the I helix, shows a double bend due to two proline residues (P360 and P364). The resulting topology orients three leucine side chains toward the active site (L358, L359 and L363). In P450 CAM [46] and P450 TERP [47], SRS 5 is a b-strand partially involved in b-sheet formation with SRS 6. In P450 BM3 [44], the first bend found in CYP2C5 is present and the C-terminal part of SRS 5 is a b-strand not involved in a b-sheet with SRS 6. The alignment of SRS 5 of CYP2C5 and the whole CYP2B family with those of CYP73A1 and CYP71Ds is not ambiguous. The two prolines and the adjacent positive charge (H365) that bind the haem propionate in CYP2C5 are conserved. This suggests that the double bend structure is present and confirms I371 as a central residue of SRS 5 in CYP73A1. If the position of the SRS relative to the haem is conserved, the phenyl side chain in the I371F mutant should stack over the haem, which would explain the complete impairment of substrate binding and the increased stability of the mutant protein. The orientation and size of SRS 6 is quite variable between the different structures and reliable alignment of K484 with the crystallized sequences is not possible. Consequently, the role of K484 could not be correlated with the mammalian structure. CYP73A1 is more closely related to CYP2C5 (47% similarity) than to any of the bacterial proteins (36%, P450 BM3 ;28%,P450 CAM ; 27%; P450 TERP ;30%,P450 eryF ), and the structure of SRS 5 seems to be conserved between CYP2C5 and CYP73A1. In order to refine our understand- ing of CYP73A1 and to gain structural information on SRS 5 topology, a new model was built based on the CYP2C5 structure exclusively (1DT6). CA was positioned in the active site of this new model, taking into account the results of the previous NMR measurements [10] and information obtained from mutagenesis (Fig. 4). In this new model, N302 easily forms a hydrogen bond with the carboxylate of CA and I371 is well positioned for hydro- phobic contact with the substrate aromatic ring. A306 was shown to be critical for substrate recognition [9]. In this new model, its methyl group is 4.8 A ˚ from the haem iron and 3.5 A ˚ from the substrate. K484 is still too far away to form a direct contact with the cinnamate. In conclusion, a combination of homology modelling and site-directed mutagenesis of CYP73A1 has identified N302 and I371 as key determinants of substrate binding and orientation for catalysis. K484 is not involved in initial substrate binding, but seems to play a significant role in catalysis, possibly by contributing to substrate reorientation during the catalytic cycle. Modification of active site residues improved affinity for substrate Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. 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