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Structural insight into the stereoselective production of PGF2α by Old Yellow Enzyme from Trypanosoma cruzi

Structural insight into the stereoselective production of PGF2α by Old Yellow Enzyme from... Abstract Old yellow enzyme (OYE) is an NADPH oxidoreductase capable of reducing a variety of compounds. It contains flavin mononucleotide (FMN) as a prosthetic group. A ternary complex structure of OYE from Trypanosoma cruzi (TcOYE) with FMN and one of the substrates, p-hydroxybenzaldehyde, shows a striking movement around the active site upon binding of the substrate. From a structural comparison of other OYE complexed with 12-oxophytodienoate, we have constructed a complex structure with another substrate, prostaglandin H2 (PGH2), to provide a proposed stereoselective reaction mechanism for the reduction of PGH2 to prostaglandin F2α by TcOYE. Old yellow enzyme, prostaglandin synthase, ternary complex, X-ray structure Prostaglandin F2α synthase (PGFS) produces prostaglandin F2α (PGF2α) from prostaglandin H2 (PGH2), and is found not only in mammals, but also in parasitic protozoa, which cause serious infections (1–6). In mammals, PGF2α overproduction causes ovarian dysfunction and miscarriage (7–9). African trypanosomiasis is characterized by miscarriage due to PGF2α overproduction correlated with parasitemia peaks (7). Therefore, the study of parasitic PGFS is of interest in order to obtain a better understanding of the pathological roles of PGF2α overproduction and to guide the development of inhibitors. Chagas’ disease, caused by (T. cruzi), affects more than 20 million people and poses a major public health and economic problem in South America (10). Anti-chagasic drugs, such as nifurtimox and 2-nitroimidazole (benznidazole) (11–14), are available, but the problems of undesirable side effects and parasite resistance of these drugs are still unsolved. Old yellow enzyme (OYE) containing a vitamin B2-derived molecule, flavin mononucleotide (FMN), essential for its catalytic reaction (15, 16), was discovered in the 1930s, and has since been identified in yeasts (17), plants (18) and bacteria (19, 20), but not in animals (Supplementary Fig. 1A and B). p-Hydroxybenzaldehyde (p-HBA) (21) is one of substrates and/or participates in a charge transfer interaction for many OYE family enzymes (Supplementary Fig. 2A). The OYE from T. cruzi (TcOYE) was identified and characterized by Kubata et al. (22), who revealed that TcOYE catalyses PGF2α synthesis as well as the reduction of many kinds of trypanocidal drugs, such as naphthoquinone and nitroheterocyclic compounds (Fig. 1). Therefore, an X-ray crystallographic study of TcOYE with these compounds should provide a structural basis for the development of novel anti-chagasic drugs. Here, we report the ternary complex structure of TcOYE/FMN/p-HBA at 2.05 Å resolution. The structure shows the striking movement of a loop covering the active site upon binding of p-HBA and reveals the recognition mechanism for a variety of compounds. Furthermore, a proposed PGH2 binding model leads us to suggest a detailed reaction mechanism from PGH2 to PGF2α by TcOYE. Fig. 1 View largeDownload slide Reaction scheme of PGH2 to PGF2α by TcOYE. FMN (cofactor of TcOYE) is reduced to FMNH2 by intravital NADPH, and the product, PGF2α, is subsequently generated by FMNH2. Fig. 1 View largeDownload slide Reaction scheme of PGH2 to PGF2α by TcOYE. FMN (cofactor of TcOYE) is reduced to FMNH2 by intravital NADPH, and the product, PGF2α, is subsequently generated by FMNH2. Materials and Methods Enzyme assay To study p-HBA inhibition of TcOYE reductase activity, various concentrations of the inhibitors were pre-incubated with an appropriate amount of NADPH and the reaction was started by the addition of TcOYE. Spectrophotometric assays were performed in a standard reaction mixture (1 ml) containing 100 mM sodium phosphate, pH 7.0, 32 µg/ml TcOYE and 500 µM NADPH. We incubated the mixture at 25°C for 2 min under aerobic conditions before adding the enzyme to initiate the reaction. After the addition of TcOYE, NADPH oxidation was monitored for 1 min by the decrease in absorbance at 340 nm. The reaction rate was determined by calculating initial rates of reaction from the known extinction coefficient of 6,220 M−1 cm−1 of NADPH. Preparation of ternary complex crystals of TcOYE/FMN/p-HBA TcOYE was overexpressed in Escherichia coli and purified according to the method as previously reported (22, 23). We first crystallized the TcOYE/FMN binary complex, and then the crystals were soaked into the cryo-protectant solution containing 80 mM p-HBA and incubated for 2 min. After incubation, the crystals were flash frozen in liquid nitrogen. The X-ray diffraction data were obtained at 2.05 Å resolution at SPring-8 beam-line BL44XU. The diffraction intensity data were processed and scaled using the program HKL2000 (24). Structure determination and refinement The structure of TcOYE was determined by molecular replacement with the program AMoRe (25) using the TcOYE structure previously solved by our group as a search model (26). Crystallographic refinement was carried out with the program CNS (27). The refinement procedure included simulated annealing, positional refinement, restrained temperature factor refinement and maximum likelihood algorithms as provided by the CNS program. Electron density maps based on the coefficients 2Fo–Fc and Fo–Fc were used to build atomic models in Coot (28). Water molecules were inserted manually and then checked by inspecting the Fo–Fc map. FMN (cofactor) and p-HBA (ligand) were refined using atomic parameters extracted from the HIC-UP server (29), and they were inserted into the clearly defined electron density model after some cycles of refinement. Results and discussion Reduction activity of TcOYE for p-HBA as a substrate p-HBA is one of substrates for enzymes belonging to the OYE family. Therefore, PGF2α synthesis by TcOYE must be competitively inhibited in the presence of p-HBA. To gain insight into the binding affinity of p-HBA to TcOYE as well as the inhibitory effect on PGH2 9,11-endoperoxide reductase activity of the enzyme, we investigated the effects of p-HBA on NADPH oxidation by TcOYE. p-HBA inhibited NADPH oxidation in a dose-dependent manner (Supplementary Fig. 2) and TcOYE exhibited 50% of the control activity in the presence of 57 μM p-HBA, indicating that this ligand was an inhibitor for the synthesis of PGF2α also in a dose-dependent manner. p-HBA binding structure of TcOYE The three-dimensional structure of the ternary complex of TcOYE/FMN/p-HBA was determined by molecular replacement using the native structure (26) as the initial model, and refined at 2.05 Å resolution with final Rcryst/Rfree values of 18.1%/22.9% (Table I). Table I. Data collection and refinement statistics.   TcOYE/FMN/p-HBA  Beam line  SPring-8 BL44XU  Space group  P21  Cell constants (Å, °)  a = 47.33, b = 119.59, c = 111.53, β = 90.35  Resolution range (Å)  50.0–2.05 (2.12–2.05)a  No. of molecules per asymmetric unit  4  VM (Å3/dalton)  1.9  Vsolv (%)  35  No. of measured reflections  297,037  No. of unique reflections  78,333  I/σ (I)  10.2  Rmerge (%)b  9.0 (33.6)a  Completeness (%)  100.0 (100.0)a  Rcryst (%)c  18.1  Rfree (%)d  22.9  R.m.s.d.'s    Bonds (Å)  0.005  Angles (°)  1.3    TcOYE/FMN/p-HBA  Beam line  SPring-8 BL44XU  Space group  P21  Cell constants (Å, °)  a = 47.33, b = 119.59, c = 111.53, β = 90.35  Resolution range (Å)  50.0–2.05 (2.12–2.05)a  No. of molecules per asymmetric unit  4  VM (Å3/dalton)  1.9  Vsolv (%)  35  No. of measured reflections  297,037  No. of unique reflections  78,333  I/σ (I)  10.2  Rmerge (%)b  9.0 (33.6)a  Completeness (%)  100.0 (100.0)a  Rcryst (%)c  18.1  Rfree (%)d  22.9  R.m.s.d.'s    Bonds (Å)  0.005  Angles (°)  1.3  aValues in parentheses are for the highest resolution shell bRmerge = Σ|I(k) − I|/ΣI(k), where I(k) is value of the kth measurement of the intensity of a reflection, I is the mean value of the intensity of that reflection and the summation is over all measurements. cRcryst = Σ||Fo| − |Fc||/Σ|Fo|, calculated from 90% of the data, which were used during the course of the refinement. dRfree = Σ||Fo| − |Fc||/Σ|Fo|, calculated from 10% of the data, which were obtained during the course of the refinement. View Large TcOYE, like other members of the OYE family, folds into a (α/β)8 barrel in which p-HBA is clearly bound at the C-terminal ends of the 8 β-strands (Fig. 2A). The structure comparison between the ternary complex of TcOYE/FMN/p-HBA and the binary complex of TcOYE/FMN provides information about the structural changes that occur upon binding of p-HBA (Supplementary Fig. 2C). While the r.m.s.d. value for Cα carbon atoms in the whole structure was calculated to be 0.34 Å, the value for Loop 1 from Lys140 to Thr157 was found to be 1.1 Å. The region from Lys140 to Tyr157 in Loop 1 was obviously shifted away from the active site of TcOYE upon binding of p-HBA. Fig. 2 View largeDownload slide Recognition of p-HBA by TcOYE. (A) Schematic representation of the ternary complex of TcOYE/FMN/p-HBA. The α-helices (in cyan) and β-sheets (in orange) are separated by loops (in light yellow). The loops shown in magenta (Loop1 and Loop2) are those related to the substrate recognition of TcOYE. FMN and p-HBA are shown as a ball-and-stick model. (B) A close-up view of the ligand binding site. A p-HBA molecule (dark blue) is positioned above the isoalloxazine ring of FMN and forms hydrogen bonds (represented in dashed lines) with 3 residues (green) of TcOYE. The p-HBA electron density is calculated at 2.05 Å and contoured at 1.0 σ. Residues shown in orange (highly conserved in OYEs) and magenta (in Loop1 unique to TcOYE) form a hydrophobic pocket surrounding the active site of TcOYE. (C) A close-up view of the active site of TcOYE (cyan) superimposed with that of OPR1 (red) by using the Cα carbon atoms of whole structures. A part of the olefinic side chain of OPDA is disordered in the structure of OPR1; however, the cyclopentane ring of OPDA is superimposed on p-HBA in the structure of TcOYE. Ligands and residues of Loop1 in both enzymes are shown with no transparency. (D) Side view of the proposed structure of TcOYE complexed with PGH2. (E) Proposed mechanism of PGH2 reduction by TcOYE. A hydride is expected to be transferred from N5 of FMN to the C9 oxygen of PGH2, whereas a proton would be donated from His195 to the C11 oxygen of PGH2. The figures of (A) and (B) were drawn by using the program MOLSCRIPT (36) and RASTER3D (37), and those of (C) and (D) were created by PyMOL (38). Fig. 2 View largeDownload slide Recognition of p-HBA by TcOYE. (A) Schematic representation of the ternary complex of TcOYE/FMN/p-HBA. The α-helices (in cyan) and β-sheets (in orange) are separated by loops (in light yellow). The loops shown in magenta (Loop1 and Loop2) are those related to the substrate recognition of TcOYE. FMN and p-HBA are shown as a ball-and-stick model. (B) A close-up view of the ligand binding site. A p-HBA molecule (dark blue) is positioned above the isoalloxazine ring of FMN and forms hydrogen bonds (represented in dashed lines) with 3 residues (green) of TcOYE. The p-HBA electron density is calculated at 2.05 Å and contoured at 1.0 σ. Residues shown in orange (highly conserved in OYEs) and magenta (in Loop1 unique to TcOYE) form a hydrophobic pocket surrounding the active site of TcOYE. (C) A close-up view of the active site of TcOYE (cyan) superimposed with that of OPR1 (red) by using the Cα carbon atoms of whole structures. A part of the olefinic side chain of OPDA is disordered in the structure of OPR1; however, the cyclopentane ring of OPDA is superimposed on p-HBA in the structure of TcOYE. Ligands and residues of Loop1 in both enzymes are shown with no transparency. (D) Side view of the proposed structure of TcOYE complexed with PGH2. (E) Proposed mechanism of PGH2 reduction by TcOYE. A hydride is expected to be transferred from N5 of FMN to the C9 oxygen of PGH2, whereas a proton would be donated from His195 to the C11 oxygen of PGH2. The figures of (A) and (B) were drawn by using the program MOLSCRIPT (36) and RASTER3D (37), and those of (C) and (D) were created by PyMOL (38). The p-HBA molecule is well-defined in the electron density of difference Fourier maps and is placed above the isoalloxazine ring of FMN via a π–π interaction (Fig. 2B). Besides, the ligand is stabilized by three hydrogen bonds: His195 and Asn198 interact with the phenolate oxygen and Tyr364 with the aldehyde carbonyl oxygen of the p-HBA molecule. His195 and Tyr364 are almost exclusively conserved among the OYE family. Asn198 is conserved as an asparagine or histidine where both Nδ atoms are involved with the binding to FMN (residues represented by green stars in Supplementary Fig. 1). Like other OYE families, the active site of TcOYE is surrounded with hydrophobic residues (30, 31), and the aromatic ring of p-HBA is stabilized in this hydrophobic pocket. Most residues forming the pocket are present in previously solved OYE members: Thr28 and Tyr200 are conserved also in other OYE members; however, Phe71 and Ile105 are conserved as tyrosine and tryptophan, respectively. It is notable that Tyr147 and Phe148 are characteristic of TcOYE. Structural comparison of TcOYE with OPR1 12-Oxophytodienoate reductase (OPR) from Lycopersicon esculentum (tomato), an OYE homologue containing a cofactor of FMN, catalyses the NADPH-dependent reduction of the cyclopentenone, 12-oxophytodienoate (OPDA) (32, 33), whose skeleton is similar to PGH2, another substrate of TcOYE (30) (Fig. 2C & Supplementary Fig. 3A). OPR1 from Lycopersicon esculentum is one of the isoforms of OPR, and shares 36% sequence identity with TcOYE (Supplementary Fig. 1). Even with the low sequence identity, the complex structure of TcOYE/FMN/p-HBA was superimposed on that of OPR1/FMN/OPDA with a r.m.s.d. value of 1.1 Å for the Cα carbon atoms (Supplementary Fig. 3B). However, the structural difference between TcOYE and OPR1 was striking at Loop1. As for the binding of OPDA, the carbonyl oxygen at C12 is hydrogen-bonded to His187(Nε) and His190(Nδ), and the carboxyl group of the α-chain interacts with Ser143(OH) in Loop1. The phenolate oxygen of p-HBA is hydrogen bonded to His195(Nε) and Asn198(Nδ) that are conserved as His187 and His190 in OPR1, respectively. Besides, Tyr364, which is hydrogen-bonded to the aldehyde carbonyl oxygen of p-HBA, is conserved as Tyr358 in OPR1 (Fig. 2C). In OPR1, Ser143 interacts with the carboxyl group of OPDA, but the corresponding residue is not conserved in Loop1 of TcOYE. Ser143 in OPR1 corresponds to Tyr147 or Phe148 in Loop1 of TcOYE (Supplementary Fig. 1A); however, both of the residues are quite close to the α-chain of OPDA (Fig. 2C). If an OPDA molecule were inserted into the active site of TcOYE, the α-chain of OPDA in its conformation in OPR1 would conflict with Tyr147 and Phe148 in Loop1 of TcOYE. Because the molecular formula of OPDA is quite similar with that of PGH2, if a PGH2 molecule binds to the active site of TcOYE as the substrate, a larger structural movement of Loop1 would also be expected to occur to avoid the steric hindrances contributed by Tyr147 and Phe148 and to bring together the α-chain of the substrate with some residue instead of Ser143 in OPR1. Notably, not only in OPR1 but also in the other solved OYEs, Loop1 was involved in binding to various ligands (31, 34); therefore, TcOYE might have evolved to recognize a PGH2 molecule as its substrate by acquiring the uniqueness and flexibility contained in the structure of Loop1. PGH2 binding model of TcOYE and proposed reaction mechanism as PGFS The initial three-dimensional coordinate and its molecular topology of the substrate PGH2 were generated by using the HIC-Up server (`Hetero-compound Information Centre, Uppsala, at URL http://alpha2.bmc.uu.se/hicup/). Because the structure formula of PGH2 is similar with that of OPDA, we then modelled the PGH2 molecule manually based on the coordinates of the OPR1/FMN/OPDA complex. The cyclopentane ring and the α-chain of PGH2 were first superimposed on those of OPDA, and a binding model of PGH2 was then constructed with energy minimization by using the conformational database potential method implemented in the refinement program CNS (27) (Fig. 2D). In CNS, the Engh and Huber force field (as described in chapter 18.3 of volume F of International Table for Crystallography, pp. 382–392, 2001) is used. As for the FMN molecule, we examined the energy minimization with both the reduced form of FMNH− and FMNH2, but the relative positions of the cyclopentane ring of PGH2 and its surroundings were not strikingly changed. Finally, the surface representation showed that PGH2 could be bound into the active site which is surrounded by many hydrophobic residues (Supplementary Fig. 3C). The hydrophobic pocket probably stabilizes the alkyl chains of PGH2 while the α-chain of PGH2 was located in the pocket formed by Tyr363, Tyr364 and Pro360 in the current model. TcOYE catalyses one- or two-electron reduction of various substrates (22). Therefore, two possible mechanisms can be envisaged for the reduction of the substrate PGH2 by a reduced flavin in TcOYE. One mechanism is the one-electron reduction including sequential electron and proton transfer. The other mechanism is the two electron reduction including hydride transfer. In case of the former, N5 or N1 of FMN could be possible as the proton or electron donor for the C9 or C11 oxygen of PGH2, respectively, because, in Fig. 2D, the distances between N5 and the C9 oxygen and between N1 and the C11 oxygen are calculated to be 3.1 and 3.3 Å, respectively. If the reaction of PGH2 to PGF2α in TcOYE occurs by two-electron reduction, it appears that a hydride transfers from N5 of FMN to the C9 oxygen of PGH2 (distance 3.1 Å), and the proton donor for the C11 oxygen is thought to be Nε of His195 (Fig. 2D). Indeed the C11 oxygen of PGH2 is located at 5.5 Å from Nε of His195 and at 3.5 Å from Nε of Asn198 in this model, but the C11 oxygen would be accessible to both His195 and Asn198 upon cleaving the peroxide after the acceptance of a hydride at the C9 oxygen of PGH2. The C9 oxygen after the reduction also could hydrogen bond to the OH atom of Thr28 (positioned at 4.1 Å from C9 oxygen in Fig. 2D). In this reaction, Nδ of His284 is located at 3.3 Å from Nε of Arg249, and at 3.4 Å from Oε of Glu193. It is likely that a triad of Glu193–His284–Arg249 is stabilized by these interactions. All residues in Fig. 2D are strongly conserved in OYE members and play an important role in the reduction of PGH2 by TcOYE (Fig. 2D). As for many other OYE families, tyrosine (corresponding to Tyr200 in TcOYE) is utilized as a proton source for substrates (35). In TcOYE, however, Tyr200 cannot provide a proton to PGH2 because the C9 and C11 oxygen atoms need to accept a hydride or proton from the same direction to maintain the stereospecificity of the biosynthesis of PGF2α by TcOYE. Hence, it is reasonable that proton transfer from His195 under the cyclopentane ring of PGH2, instead of Tyr200, would occur to achieve the stereoselective production of PGF2α (Fig. 2D and E). We believe that the structural analysis of the ternary complex of TcOYE/FMN/p-HBA as it primarily relates to substrate binding of PGH2 may lead to the development of heroic anti-chagasic drugs. Funding Grants-in-Aid for Scientific Research (No. 22550152) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the PRESTO project, Japan Science and Technology Agency, the National Project on Protein Structural and Functional Analyses, Japan (to T.I.); High-quality Protein Crystal Growth Experiment on JEM promoted by JAXA and Osaka City (to Y.U.). Conflict of interest None declared. Acknowledgements The authors express their appreciation to Professor B. K. Kubata, Biosciences Eastern and Central Africa, and Dr Z. Kabututu, Osaka Bioscience Institute, for their early support of protein expression. The authors are grateful to M. Yoshimura, M. Yamashita, E. Yamashita and A. Nakagawa for their kind support during data collection at SPring-8 beamline 44XU. Abbreviations Abbreviations FMN flavin mononucleotide OPR 12-oxophytodienoate reductase OPDA 12-oxophytodienoate OYE old yellow enzyme PG prostaglandin PGFS prostaglandin F2α synthase p-HBA p-hydroxybenzaldehyde T. cruzi Trypanosoma cruzi TcOYE OYE from T. cruzi References 1 Kilunga Kubata B,  Eguchi N,  Urade Y,  Yamashita K,  Mitamura T,  Tai K,  Hayaishi O,  Horii T.  Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans,  J. Exp. Med. ,  1998, vol.  188 (pg.  1197- 1202) Google Scholar CrossRef Search ADS PubMed  2 Kubata BK,  Duszenko M,  Kabututu Z,  Rawer M,  Szallies A,  Fujimori K,  Inui T,  Nozaki T,  Yamashita K,  Horii T,  Urade Y,  Hayaishi O.  Identification of a novel prostaglandin F2α synthase in Trypanosoma brucei,  J. Exp. 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Structural insight into the stereoselective production of PGF2α by Old Yellow Enzyme from Trypanosoma cruzi

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Oxford University Press
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© The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
DOI
10.1093/jb/mvr096
pmid
21840922
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Abstract

Abstract Old yellow enzyme (OYE) is an NADPH oxidoreductase capable of reducing a variety of compounds. It contains flavin mononucleotide (FMN) as a prosthetic group. A ternary complex structure of OYE from Trypanosoma cruzi (TcOYE) with FMN and one of the substrates, p-hydroxybenzaldehyde, shows a striking movement around the active site upon binding of the substrate. From a structural comparison of other OYE complexed with 12-oxophytodienoate, we have constructed a complex structure with another substrate, prostaglandin H2 (PGH2), to provide a proposed stereoselective reaction mechanism for the reduction of PGH2 to prostaglandin F2α by TcOYE. Old yellow enzyme, prostaglandin synthase, ternary complex, X-ray structure Prostaglandin F2α synthase (PGFS) produces prostaglandin F2α (PGF2α) from prostaglandin H2 (PGH2), and is found not only in mammals, but also in parasitic protozoa, which cause serious infections (1–6). In mammals, PGF2α overproduction causes ovarian dysfunction and miscarriage (7–9). African trypanosomiasis is characterized by miscarriage due to PGF2α overproduction correlated with parasitemia peaks (7). Therefore, the study of parasitic PGFS is of interest in order to obtain a better understanding of the pathological roles of PGF2α overproduction and to guide the development of inhibitors. Chagas’ disease, caused by (T. cruzi), affects more than 20 million people and poses a major public health and economic problem in South America (10). Anti-chagasic drugs, such as nifurtimox and 2-nitroimidazole (benznidazole) (11–14), are available, but the problems of undesirable side effects and parasite resistance of these drugs are still unsolved. Old yellow enzyme (OYE) containing a vitamin B2-derived molecule, flavin mononucleotide (FMN), essential for its catalytic reaction (15, 16), was discovered in the 1930s, and has since been identified in yeasts (17), plants (18) and bacteria (19, 20), but not in animals (Supplementary Fig. 1A and B). p-Hydroxybenzaldehyde (p-HBA) (21) is one of substrates and/or participates in a charge transfer interaction for many OYE family enzymes (Supplementary Fig. 2A). The OYE from T. cruzi (TcOYE) was identified and characterized by Kubata et al. (22), who revealed that TcOYE catalyses PGF2α synthesis as well as the reduction of many kinds of trypanocidal drugs, such as naphthoquinone and nitroheterocyclic compounds (Fig. 1). Therefore, an X-ray crystallographic study of TcOYE with these compounds should provide a structural basis for the development of novel anti-chagasic drugs. Here, we report the ternary complex structure of TcOYE/FMN/p-HBA at 2.05 Å resolution. The structure shows the striking movement of a loop covering the active site upon binding of p-HBA and reveals the recognition mechanism for a variety of compounds. Furthermore, a proposed PGH2 binding model leads us to suggest a detailed reaction mechanism from PGH2 to PGF2α by TcOYE. Fig. 1 View largeDownload slide Reaction scheme of PGH2 to PGF2α by TcOYE. FMN (cofactor of TcOYE) is reduced to FMNH2 by intravital NADPH, and the product, PGF2α, is subsequently generated by FMNH2. Fig. 1 View largeDownload slide Reaction scheme of PGH2 to PGF2α by TcOYE. FMN (cofactor of TcOYE) is reduced to FMNH2 by intravital NADPH, and the product, PGF2α, is subsequently generated by FMNH2. Materials and Methods Enzyme assay To study p-HBA inhibition of TcOYE reductase activity, various concentrations of the inhibitors were pre-incubated with an appropriate amount of NADPH and the reaction was started by the addition of TcOYE. Spectrophotometric assays were performed in a standard reaction mixture (1 ml) containing 100 mM sodium phosphate, pH 7.0, 32 µg/ml TcOYE and 500 µM NADPH. We incubated the mixture at 25°C for 2 min under aerobic conditions before adding the enzyme to initiate the reaction. After the addition of TcOYE, NADPH oxidation was monitored for 1 min by the decrease in absorbance at 340 nm. The reaction rate was determined by calculating initial rates of reaction from the known extinction coefficient of 6,220 M−1 cm−1 of NADPH. Preparation of ternary complex crystals of TcOYE/FMN/p-HBA TcOYE was overexpressed in Escherichia coli and purified according to the method as previously reported (22, 23). We first crystallized the TcOYE/FMN binary complex, and then the crystals were soaked into the cryo-protectant solution containing 80 mM p-HBA and incubated for 2 min. After incubation, the crystals were flash frozen in liquid nitrogen. The X-ray diffraction data were obtained at 2.05 Å resolution at SPring-8 beam-line BL44XU. The diffraction intensity data were processed and scaled using the program HKL2000 (24). Structure determination and refinement The structure of TcOYE was determined by molecular replacement with the program AMoRe (25) using the TcOYE structure previously solved by our group as a search model (26). Crystallographic refinement was carried out with the program CNS (27). The refinement procedure included simulated annealing, positional refinement, restrained temperature factor refinement and maximum likelihood algorithms as provided by the CNS program. Electron density maps based on the coefficients 2Fo–Fc and Fo–Fc were used to build atomic models in Coot (28). Water molecules were inserted manually and then checked by inspecting the Fo–Fc map. FMN (cofactor) and p-HBA (ligand) were refined using atomic parameters extracted from the HIC-UP server (29), and they were inserted into the clearly defined electron density model after some cycles of refinement. Results and discussion Reduction activity of TcOYE for p-HBA as a substrate p-HBA is one of substrates for enzymes belonging to the OYE family. Therefore, PGF2α synthesis by TcOYE must be competitively inhibited in the presence of p-HBA. To gain insight into the binding affinity of p-HBA to TcOYE as well as the inhibitory effect on PGH2 9,11-endoperoxide reductase activity of the enzyme, we investigated the effects of p-HBA on NADPH oxidation by TcOYE. p-HBA inhibited NADPH oxidation in a dose-dependent manner (Supplementary Fig. 2) and TcOYE exhibited 50% of the control activity in the presence of 57 μM p-HBA, indicating that this ligand was an inhibitor for the synthesis of PGF2α also in a dose-dependent manner. p-HBA binding structure of TcOYE The three-dimensional structure of the ternary complex of TcOYE/FMN/p-HBA was determined by molecular replacement using the native structure (26) as the initial model, and refined at 2.05 Å resolution with final Rcryst/Rfree values of 18.1%/22.9% (Table I). Table I. Data collection and refinement statistics.   TcOYE/FMN/p-HBA  Beam line  SPring-8 BL44XU  Space group  P21  Cell constants (Å, °)  a = 47.33, b = 119.59, c = 111.53, β = 90.35  Resolution range (Å)  50.0–2.05 (2.12–2.05)a  No. of molecules per asymmetric unit  4  VM (Å3/dalton)  1.9  Vsolv (%)  35  No. of measured reflections  297,037  No. of unique reflections  78,333  I/σ (I)  10.2  Rmerge (%)b  9.0 (33.6)a  Completeness (%)  100.0 (100.0)a  Rcryst (%)c  18.1  Rfree (%)d  22.9  R.m.s.d.'s    Bonds (Å)  0.005  Angles (°)  1.3    TcOYE/FMN/p-HBA  Beam line  SPring-8 BL44XU  Space group  P21  Cell constants (Å, °)  a = 47.33, b = 119.59, c = 111.53, β = 90.35  Resolution range (Å)  50.0–2.05 (2.12–2.05)a  No. of molecules per asymmetric unit  4  VM (Å3/dalton)  1.9  Vsolv (%)  35  No. of measured reflections  297,037  No. of unique reflections  78,333  I/σ (I)  10.2  Rmerge (%)b  9.0 (33.6)a  Completeness (%)  100.0 (100.0)a  Rcryst (%)c  18.1  Rfree (%)d  22.9  R.m.s.d.'s    Bonds (Å)  0.005  Angles (°)  1.3  aValues in parentheses are for the highest resolution shell bRmerge = Σ|I(k) − I|/ΣI(k), where I(k) is value of the kth measurement of the intensity of a reflection, I is the mean value of the intensity of that reflection and the summation is over all measurements. cRcryst = Σ||Fo| − |Fc||/Σ|Fo|, calculated from 90% of the data, which were used during the course of the refinement. dRfree = Σ||Fo| − |Fc||/Σ|Fo|, calculated from 10% of the data, which were obtained during the course of the refinement. View Large TcOYE, like other members of the OYE family, folds into a (α/β)8 barrel in which p-HBA is clearly bound at the C-terminal ends of the 8 β-strands (Fig. 2A). The structure comparison between the ternary complex of TcOYE/FMN/p-HBA and the binary complex of TcOYE/FMN provides information about the structural changes that occur upon binding of p-HBA (Supplementary Fig. 2C). While the r.m.s.d. value for Cα carbon atoms in the whole structure was calculated to be 0.34 Å, the value for Loop 1 from Lys140 to Thr157 was found to be 1.1 Å. The region from Lys140 to Tyr157 in Loop 1 was obviously shifted away from the active site of TcOYE upon binding of p-HBA. Fig. 2 View largeDownload slide Recognition of p-HBA by TcOYE. (A) Schematic representation of the ternary complex of TcOYE/FMN/p-HBA. The α-helices (in cyan) and β-sheets (in orange) are separated by loops (in light yellow). The loops shown in magenta (Loop1 and Loop2) are those related to the substrate recognition of TcOYE. FMN and p-HBA are shown as a ball-and-stick model. (B) A close-up view of the ligand binding site. A p-HBA molecule (dark blue) is positioned above the isoalloxazine ring of FMN and forms hydrogen bonds (represented in dashed lines) with 3 residues (green) of TcOYE. The p-HBA electron density is calculated at 2.05 Å and contoured at 1.0 σ. Residues shown in orange (highly conserved in OYEs) and magenta (in Loop1 unique to TcOYE) form a hydrophobic pocket surrounding the active site of TcOYE. (C) A close-up view of the active site of TcOYE (cyan) superimposed with that of OPR1 (red) by using the Cα carbon atoms of whole structures. A part of the olefinic side chain of OPDA is disordered in the structure of OPR1; however, the cyclopentane ring of OPDA is superimposed on p-HBA in the structure of TcOYE. Ligands and residues of Loop1 in both enzymes are shown with no transparency. (D) Side view of the proposed structure of TcOYE complexed with PGH2. (E) Proposed mechanism of PGH2 reduction by TcOYE. A hydride is expected to be transferred from N5 of FMN to the C9 oxygen of PGH2, whereas a proton would be donated from His195 to the C11 oxygen of PGH2. The figures of (A) and (B) were drawn by using the program MOLSCRIPT (36) and RASTER3D (37), and those of (C) and (D) were created by PyMOL (38). Fig. 2 View largeDownload slide Recognition of p-HBA by TcOYE. (A) Schematic representation of the ternary complex of TcOYE/FMN/p-HBA. The α-helices (in cyan) and β-sheets (in orange) are separated by loops (in light yellow). The loops shown in magenta (Loop1 and Loop2) are those related to the substrate recognition of TcOYE. FMN and p-HBA are shown as a ball-and-stick model. (B) A close-up view of the ligand binding site. A p-HBA molecule (dark blue) is positioned above the isoalloxazine ring of FMN and forms hydrogen bonds (represented in dashed lines) with 3 residues (green) of TcOYE. The p-HBA electron density is calculated at 2.05 Å and contoured at 1.0 σ. Residues shown in orange (highly conserved in OYEs) and magenta (in Loop1 unique to TcOYE) form a hydrophobic pocket surrounding the active site of TcOYE. (C) A close-up view of the active site of TcOYE (cyan) superimposed with that of OPR1 (red) by using the Cα carbon atoms of whole structures. A part of the olefinic side chain of OPDA is disordered in the structure of OPR1; however, the cyclopentane ring of OPDA is superimposed on p-HBA in the structure of TcOYE. Ligands and residues of Loop1 in both enzymes are shown with no transparency. (D) Side view of the proposed structure of TcOYE complexed with PGH2. (E) Proposed mechanism of PGH2 reduction by TcOYE. A hydride is expected to be transferred from N5 of FMN to the C9 oxygen of PGH2, whereas a proton would be donated from His195 to the C11 oxygen of PGH2. The figures of (A) and (B) were drawn by using the program MOLSCRIPT (36) and RASTER3D (37), and those of (C) and (D) were created by PyMOL (38). The p-HBA molecule is well-defined in the electron density of difference Fourier maps and is placed above the isoalloxazine ring of FMN via a π–π interaction (Fig. 2B). Besides, the ligand is stabilized by three hydrogen bonds: His195 and Asn198 interact with the phenolate oxygen and Tyr364 with the aldehyde carbonyl oxygen of the p-HBA molecule. His195 and Tyr364 are almost exclusively conserved among the OYE family. Asn198 is conserved as an asparagine or histidine where both Nδ atoms are involved with the binding to FMN (residues represented by green stars in Supplementary Fig. 1). Like other OYE families, the active site of TcOYE is surrounded with hydrophobic residues (30, 31), and the aromatic ring of p-HBA is stabilized in this hydrophobic pocket. Most residues forming the pocket are present in previously solved OYE members: Thr28 and Tyr200 are conserved also in other OYE members; however, Phe71 and Ile105 are conserved as tyrosine and tryptophan, respectively. It is notable that Tyr147 and Phe148 are characteristic of TcOYE. Structural comparison of TcOYE with OPR1 12-Oxophytodienoate reductase (OPR) from Lycopersicon esculentum (tomato), an OYE homologue containing a cofactor of FMN, catalyses the NADPH-dependent reduction of the cyclopentenone, 12-oxophytodienoate (OPDA) (32, 33), whose skeleton is similar to PGH2, another substrate of TcOYE (30) (Fig. 2C & Supplementary Fig. 3A). OPR1 from Lycopersicon esculentum is one of the isoforms of OPR, and shares 36% sequence identity with TcOYE (Supplementary Fig. 1). Even with the low sequence identity, the complex structure of TcOYE/FMN/p-HBA was superimposed on that of OPR1/FMN/OPDA with a r.m.s.d. value of 1.1 Å for the Cα carbon atoms (Supplementary Fig. 3B). However, the structural difference between TcOYE and OPR1 was striking at Loop1. As for the binding of OPDA, the carbonyl oxygen at C12 is hydrogen-bonded to His187(Nε) and His190(Nδ), and the carboxyl group of the α-chain interacts with Ser143(OH) in Loop1. The phenolate oxygen of p-HBA is hydrogen bonded to His195(Nε) and Asn198(Nδ) that are conserved as His187 and His190 in OPR1, respectively. Besides, Tyr364, which is hydrogen-bonded to the aldehyde carbonyl oxygen of p-HBA, is conserved as Tyr358 in OPR1 (Fig. 2C). In OPR1, Ser143 interacts with the carboxyl group of OPDA, but the corresponding residue is not conserved in Loop1 of TcOYE. Ser143 in OPR1 corresponds to Tyr147 or Phe148 in Loop1 of TcOYE (Supplementary Fig. 1A); however, both of the residues are quite close to the α-chain of OPDA (Fig. 2C). If an OPDA molecule were inserted into the active site of TcOYE, the α-chain of OPDA in its conformation in OPR1 would conflict with Tyr147 and Phe148 in Loop1 of TcOYE. Because the molecular formula of OPDA is quite similar with that of PGH2, if a PGH2 molecule binds to the active site of TcOYE as the substrate, a larger structural movement of Loop1 would also be expected to occur to avoid the steric hindrances contributed by Tyr147 and Phe148 and to bring together the α-chain of the substrate with some residue instead of Ser143 in OPR1. Notably, not only in OPR1 but also in the other solved OYEs, Loop1 was involved in binding to various ligands (31, 34); therefore, TcOYE might have evolved to recognize a PGH2 molecule as its substrate by acquiring the uniqueness and flexibility contained in the structure of Loop1. PGH2 binding model of TcOYE and proposed reaction mechanism as PGFS The initial three-dimensional coordinate and its molecular topology of the substrate PGH2 were generated by using the HIC-Up server (`Hetero-compound Information Centre, Uppsala, at URL http://alpha2.bmc.uu.se/hicup/). Because the structure formula of PGH2 is similar with that of OPDA, we then modelled the PGH2 molecule manually based on the coordinates of the OPR1/FMN/OPDA complex. The cyclopentane ring and the α-chain of PGH2 were first superimposed on those of OPDA, and a binding model of PGH2 was then constructed with energy minimization by using the conformational database potential method implemented in the refinement program CNS (27) (Fig. 2D). In CNS, the Engh and Huber force field (as described in chapter 18.3 of volume F of International Table for Crystallography, pp. 382–392, 2001) is used. As for the FMN molecule, we examined the energy minimization with both the reduced form of FMNH− and FMNH2, but the relative positions of the cyclopentane ring of PGH2 and its surroundings were not strikingly changed. Finally, the surface representation showed that PGH2 could be bound into the active site which is surrounded by many hydrophobic residues (Supplementary Fig. 3C). The hydrophobic pocket probably stabilizes the alkyl chains of PGH2 while the α-chain of PGH2 was located in the pocket formed by Tyr363, Tyr364 and Pro360 in the current model. TcOYE catalyses one- or two-electron reduction of various substrates (22). Therefore, two possible mechanisms can be envisaged for the reduction of the substrate PGH2 by a reduced flavin in TcOYE. One mechanism is the one-electron reduction including sequential electron and proton transfer. The other mechanism is the two electron reduction including hydride transfer. In case of the former, N5 or N1 of FMN could be possible as the proton or electron donor for the C9 or C11 oxygen of PGH2, respectively, because, in Fig. 2D, the distances between N5 and the C9 oxygen and between N1 and the C11 oxygen are calculated to be 3.1 and 3.3 Å, respectively. If the reaction of PGH2 to PGF2α in TcOYE occurs by two-electron reduction, it appears that a hydride transfers from N5 of FMN to the C9 oxygen of PGH2 (distance 3.1 Å), and the proton donor for the C11 oxygen is thought to be Nε of His195 (Fig. 2D). Indeed the C11 oxygen of PGH2 is located at 5.5 Å from Nε of His195 and at 3.5 Å from Nε of Asn198 in this model, but the C11 oxygen would be accessible to both His195 and Asn198 upon cleaving the peroxide after the acceptance of a hydride at the C9 oxygen of PGH2. The C9 oxygen after the reduction also could hydrogen bond to the OH atom of Thr28 (positioned at 4.1 Å from C9 oxygen in Fig. 2D). In this reaction, Nδ of His284 is located at 3.3 Å from Nε of Arg249, and at 3.4 Å from Oε of Glu193. It is likely that a triad of Glu193–His284–Arg249 is stabilized by these interactions. All residues in Fig. 2D are strongly conserved in OYE members and play an important role in the reduction of PGH2 by TcOYE (Fig. 2D). As for many other OYE families, tyrosine (corresponding to Tyr200 in TcOYE) is utilized as a proton source for substrates (35). In TcOYE, however, Tyr200 cannot provide a proton to PGH2 because the C9 and C11 oxygen atoms need to accept a hydride or proton from the same direction to maintain the stereospecificity of the biosynthesis of PGF2α by TcOYE. Hence, it is reasonable that proton transfer from His195 under the cyclopentane ring of PGH2, instead of Tyr200, would occur to achieve the stereoselective production of PGF2α (Fig. 2D and E). We believe that the structural analysis of the ternary complex of TcOYE/FMN/p-HBA as it primarily relates to substrate binding of PGH2 may lead to the development of heroic anti-chagasic drugs. Funding Grants-in-Aid for Scientific Research (No. 22550152) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the PRESTO project, Japan Science and Technology Agency, the National Project on Protein Structural and Functional Analyses, Japan (to T.I.); High-quality Protein Crystal Growth Experiment on JEM promoted by JAXA and Osaka City (to Y.U.). Conflict of interest None declared. Acknowledgements The authors express their appreciation to Professor B. K. Kubata, Biosciences Eastern and Central Africa, and Dr Z. Kabututu, Osaka Bioscience Institute, for their early support of protein expression. The authors are grateful to M. Yoshimura, M. Yamashita, E. Yamashita and A. Nakagawa for their kind support during data collection at SPring-8 beamline 44XU. Abbreviations Abbreviations FMN flavin mononucleotide OPR 12-oxophytodienoate reductase OPDA 12-oxophytodienoate OYE old yellow enzyme PG prostaglandin PGFS prostaglandin F2α synthase p-HBA p-hydroxybenzaldehyde T. cruzi Trypanosoma cruzi TcOYE OYE from T. cruzi References 1 Kilunga Kubata B,  Eguchi N,  Urade Y,  Yamashita K,  Mitamura T,  Tai K,  Hayaishi O,  Horii T.  Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans,  J. Exp. Med. ,  1998, vol.  188 (pg.  1197- 1202) Google Scholar CrossRef Search ADS PubMed  2 Kubata BK,  Duszenko M,  Kabututu Z,  Rawer M,  Szallies A,  Fujimori K,  Inui T,  Nozaki T,  Yamashita K,  Horii T,  Urade Y,  Hayaishi O.  Identification of a novel prostaglandin F2α synthase in Trypanosoma brucei,  J. Exp. 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Journal

The Journal of BiochemistryOxford University Press

Published: Aug 12, 2011

Keywords: Old yellow enzyme prostaglandin synthase ternary complex X-ray structure

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