Structure, Vol. 9, 419–429, May, 2001, 2001 Elsevier Science Ltd. All rights reserved.
PII S0969-2126(01)00602-5
X-Ray Structure of 12-Oxophytodienoate Reductase 1 Provides Structural Insight into Substrate Binding and Specificity within the Family of OYE Constanze Breithaupt,1,3 Jochen Strassner,2 Ulrike Breitinger,1 Robert Huber,1 Peter Macheroux,2 Andreas Schaller,2 and Tim Clausen1,3 1 Max-Planck-Institut fu¨r Biochemie Abteilung Strukturforschung Am Klopferspitz 18a D-82152 Martinsried Germany 2 Eidgeno¨ssische Technische Hochschule Institut fu¨r Pflanzenwissenschaften Universita¨tstr. 2 CH-8092 Zu¨rich Switzerland
Introduction
Summary Background: 12-Oxophytodienoate reductase (OPR) is a flavin mononucleotide (FMN)-dependent oxidoreductase in plants that belongs to the family of Old Yellow Enzyme (OYE). It was initially characterized as an enzyme involved in the biosynthesis of the plant hormone jasmonic acid, where it catalyzes the reduction of the cyclic fatty acid derivative 9S,13S-12-oxophytodienoate (9S,13S-OPDA) to 1S,2S-3-oxo-2(2⬘[Z]-pentenyl)-cyclopentane-1-octanoate. Several isozymes of OPR are now known that show different stereoselectivities with regard to the four stereoisomers of OPDA. Results: Here, we report the high-resolution crystal structure of OPR1 from Lycopersicon esculentum and its complex structures with the substrate 9R,13R-OPDA and with polyethylene glycol 400. OPR1 crystallizes as a monomer and folds into a (␣)8 barrel with an overall structure similar to OYE. The cyclopentenone ring of 9R,13R-OPDA is stacked above the flavin and activated by two hydrogen bonds to His187 and His190. The olefinic bond is properly positioned for hydride transfer from the FMN N(5) and proton transfer from Tyr192 to C and C␣, respectively. Comparison of the OPR1 and OYE structures reveals striking differences in the loops responsible for binding 9R,13R-OPDA in OPR1. Conclusions: Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that ␣,-unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9R,13R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9R,13R-OPDA or a structurally related oxylipin as natural substrate of OPR1. 3 Correspondence:
[email protected] biochem.mpg.de (T.C.)
(C.B.),
clausen@
Old Yellow Enzyme (OYE; EC 1.6.99.1), originally purified from brewer’s bottom yeast more than 60 years ago, was the first enzyme found to require a small organic molecule—flavin mononucleotide (FMN)—for catalytic function [1, 2]. Despite extensive biochemical and spectroscopic characterization, elucidation of the threedimensional structure [3], and subsequent detailed mechanistic studies [4–7], the physiological function of OYE still remains unknown. During the last years, several FMN-linked oxidoreductases, related to OYE, have been isolated and cloned from bacteria, fungi, and plants [8–14]. Using NAD(P)H as reductant, they reduce ␣,-unsaturated ketones and aldehydes as well as aromatic nitro compounds and nitrate esters. 12-Oxophytodienoate reductase (OPR; EC 1.3.1.42) was initially characterized from corn in 1986 and is involved in the biosynthesis of jasmonic acid (JA), where it catalyzes the NADPH-dependent reduction of the cyclopentenone 12-oxophytodienoate (OPDA) to 3-oxo2(2⬘[Z]-pentenyl)-cyclopentane-1-octanoate (OPC-8:0) ([15], reviewed in [16]). JA is known to influence numerous developmental processes as growth regulator in plants, and, furthermore, it acts as an important signal molecule in plant defense against herbivores and pathogens (reviewed in [17]). Molecular cloning and sequence determination of OPR from Arabidopsis thaliana revealed the surprising similarity to OYE (39% sequence identity) and thus made OPR the only member of the OYE family with known physiological function [13] and the only one from higher eukaryotes. In the meantime, several isozymes of OPR have been discovered in A. thaliana and Lycopersicon esculentum that differ in their preference for the four stereoisomers of OPDA. OPR3, recently cloned from A. thaliana [18], effectively reduces the JA precursor 9S,13S-OPDA [cis(⫹)-OPDA], while OPR1 from A. thaliana strongly prefers the enantiomeric 9R,13R-OPDA and to a lesser extent the diastereomeric 9S,13R-OPDA [19]. The latter compound can be formed by isomerization of 9S,13S-OPDA via the enol form, while 9R,13R-OPDA was shown to be generated by nonenzymatic, spontaneous cyclization of the OPDA precursor 12,13(S)-epoxylinolenic acid in vitro [20]. Therefore, it has been assumed that 9R,13R-OPDA may be formed the same way in vivo. OPR1 could then serve to remove these undesired stereoisomers [19]. Alternatively, the 9R,13R stereoisomer could play an independent or additional role in plant signaling. Here, we report the first crystal structure of an OPR, OPR1 from L. esculentum, in its uncomplexed form, as well as the structures of complexes of OPR1 with 9R,13R-OPDA and polyethylene glycol 400 (PEG400). The active site is analyzed with respect to the observed substrate selectivity and the mechanism of substrate reduction proposed for OYE. The structures of OPR1 Key words: flavoenzyme; jasmonic acid; octadecanoids; OPR; OYE; plant defense
Structure 420
Table 1. Data Collection and Refinement Statistics Data Set
PEG400-OPR1
OPDA-OPR1
OPR1
Number of observed reflections Number of unique reflections Limiting resolution (A˚) Completeness of data (%)a Rmerge (%)b I/(I) (I) Rcryst/Rfree (%)c Number of atoms (protein/cofactor/ligand) Number of water molecules Rmsd (bonds [A˚]/angles [⬚]/bonded Bs [A˚2]d) Mean temperature factors (protein/cofactor/ligand/solvent)
101,396 66,928 1.9 91.8 (88.5) 4.9 (34.3) 13.1 (1.9) 18.2/22.2 5,640/62/56 483 0.007/1.36/2.31 24.9/19.2/48.0/33.1
99,966 54,064 2.0 83.6 (54.6) 4.3 (14.9) 14.6 (2.9) 19.8/23.4 5,576/62/36 317 0.009/1.44/2.42 36.4/31.3/44.9/40.7
73,998 40,681 2.3 96.6 (89.6) 3.8 (13.6) 17.1 (4.0) 19.7/23.7 5,568/62/— 217 0.008/1.39/2.42 37.5/33.6/—/39.7
a Values in parenthesis correspond to the highest resolution shell between 1.90 A˚ and 1.96 A˚ (PEG400-OPR1), 2.03 A˚ and 2.00 A˚ (OPDAOPR1), and 2.34 A˚ and 2.30 A˚ (OPR1). b Rmerge ⫽ ⌺h⌺i|Ii(h) ⫺ ⬍I(h)⬎|/⌺h⌺iIi(h). c Rcryst ⫽ ⌺h||Fo(h)| ⫺ |Fc(h)||/⌺h|Fo(h)|. Rfree was determined from 5% of the data that were not used for refinement. d Rmsd of bonded Bs: rmsd of temperature factors of bonded atoms
and OYE are compared, and the different substrate binding sites are discussed. Results and Discussion Overall Structure The structures of the PEG400-OPR1 complex, the PEG400-free, and the OPDA complexed enzyme were solved with resolution limits of 1.9 A˚, 2.3 A˚, and 2.0 A˚, respectively, and final Rcryst/Rfree values of 18.2%/22.2%, 19.7%/23.7%, and 19.8%/23.4% (Table 1). All residues are found in most-favored or additionally allowed regions of the Ramachandran plot (in most-favored regions: 88.8%, 87.4%, and 88.6% residues, respectively). OPR1 consists of a single 376 residue domain that folds into an eight-stranded parallel ␣ barrel with approximate dimensions 55 ⫻ 44 ⫻ 45 A˚3 (Figure 1). The overall structure of OPR1 closely resembles the structure of OYE [3] (Figure 2); 315 residues can be superimposed with an rmsd of 1.1 A˚, which is within the expected range of structural similarity, given a sequence identity of 40% between OPR1 and OYE [21]. Apart from OYE, the closest structural neighbor of OPR1 is trimethylamine dehydrogenase [22], with 263 equivalent residues and an rmsd of 1.5 A˚, followed by the flavoproteins glycolate oxidase [23], flavocytochrome b2 [24], and dihydroorotate dehydrogenase [25], and the NADHdependent inosine monophosphate dehydrogenase [26, 27] (rmsd of 1.8 A˚, with 135–143 corresponding residues). All structures exhibit a short  hairpin prior to helix ␣1 that closes the barrel at the N terminus and an additional helix ␣B in L8, the loop following strand 8 (Figure 1c). Residues of helix ␣B and of the strands 7 and 8 form a conserved phosphate binding motif that exists in various (␣)8 barrel enzymes [28] and serves to bind the phosphate group of FMN in the flavoproteins mentioned above and the phosphate of the substrate analog 6-chloropurine-riboside-5⬘-monophosphate in inosine monophosphate dehydrogenase, respectively. Except for L8, the loops at the carboxy-terminal end of the  strands adopt very different conformations in the (␣)8 barrel flavoproteins. The extra helix ␣A, inserted after
strand 4, is involved in substrate binding of OPR1 and is only present in OPR1, OYE, and trimethylamine dehydrogenase. The cave formed by ␣A and the carboxyl termini of strands 3 and 4 contains the active site residues Gln110, Trp112, Tyr192, His187, and His190. Similarly to ␣B in the phosphate binding site, the positive end of the macrodipole of ␣A points directly toward the negatively polarized carbonyl oxygen of the substrate and thus supports substrate binding and activation for hydride transfer. Consistent with the importance of these two motifs for cofactor binding and enzymatic function, they belong to the most highly conserved regions in the sequences of the OYE family. The greatest structural differences between OPR1 and OYE are found for the loops L3 and L6 (Figure 2). In OPR1, L3 is composed of one parallel and one antiparallel  sheet and adopts a cross-shaped conformation entirely different from the conformation of L3 in OYE with ␣␣ structure. By bending down deeply toward the FMN, the antiparallel  sheet is actively involved in the formation of the active site cavity, while the corresponding ␣ helix of OYE is located at a distance of 24 A˚ to the FMN isoalloxazine ring. By contrast, L6 forms part of the active site in OYE, while L6 of OPR1 is 9 residues shorter and projects away from the active site. Both loops exhibit pronounced mobility in OPR1, as indicated by the elevated temperature factor of L3 (4.4 A˚2 above the average 24.9 A˚2 for the whole protein) and by the disorder of residues 283 to 288 in L6. Structure refinement revealed the reason for the absolute necessity of the additive PEG400 in the crystallization buffer. PEG400 is bound in the active site, thereby stabilizing the protein conformation by numerous van der Waals contacts with the hydrophobic interior of the active site channel (Figure 1b). The PEG400 molecule (C18H38O10) is visible in full-length as continuous electron density in the active site. Rigidity is also imposed onto L6 that is restrained from moving toward the active site by one end of PEG400 occupying this region. In the PEG400-free structure, 4 additional residues in L6 become disordered. Residues 140–150 of L3 move away from the active site, showing a maximum deviation in C␣ positions of 1.4 A˚ compared to the PEG400-OPR1 structure.
Crystal Structure of OPR1 421
Figure 1. Overall Structure of OPR1 (a) Ribbon diagram of OPR1. The FMN cofactor and the bound PEG400 are depicted as stick models. (b) Surface representation of OPR1 with bound FMN and PEG400. The electrostatic potential was calculated with GRASP, using charges of Weiner et al. [66]. The figure shows the active site tunnel formed by the hydrophobic  strands D and E of loop L3. (c) Topology of OPR1. Colors of strands and helices correspond to (a).
In contrast to OYE, no significant interaction exists between the OPR1 protomers in the crystal, which is consistent with earlier findings that OPR1 from A. thaliana is a monomer in solution [30]. The helices ␣5 and ␣6 that form most of the dimer interface in OYE exhibit very weak sequence similarity and are of different length in OPR1 and OYE. These differences may thus account for the two observed oligomerization states.
FMN Binding Site The FMN cofactor is embedded inside the barrel interacting with the loops L7 and L8 and the carboxyl termini of all  strands that form the barrel except 4 and 6. The tight binding of the cofactor can also be inferred from the low average temperature factor of 19.5 A˚2 for FMN compared to 24.9 A˚2 for the protein atoms. Due to the proximity of 1, the re face of FMN is com-
Figure 2. Stereo View of the Overlaid C␣ Backbone of OPR1 and OYE OPR1 and OYE are depicted in green and yellow, respectively. The PEG400 molecule and the FMN cofactor of OPR1 are shown as stick models; termini and selected secondary structural elements are indicated. Residues are numbered according to the OPR1 sequence. The structural alignment was performed with the program LSQMAN [67].
Structure 422
Figure 3. FMN Binding Site of OPR1 (a) Stereo plot of the final electron density of the FMN cofactor and the FMN binding residues. The 2Fo-Fc map is calculated at 1.9 A˚ and contoured at 1.0 . (b) Schematic representation of the FMN hydrogen bonding network (FMN, red; solvent, blue; protein residues, green).
pletely buried, whereas C4 and the pyrazine ring are accessible to solvent from the si face. Moreover, parts of the ribityl chain and atoms C8 to C10, including the C8 methyl group, are exposed to solvent in OPR1 (Figure 1b). In OYE, these positions are shielded by interaction with residues of L6 that have no equivalent in OPR1. While the dimethylbenzene moiety of FMN is surrounded by the hydrophobic residues Leu36 and Phe357, the polar atoms of FMN are involved in an extensive hydrogen bonding network with the protein (Figure 3). The phosphate group interacts with main chain atoms of residues 308–310 and 330–332, while the ribityl oxygens hydrogen bond to Arg239, Pro35, and two ordered water molecules. N(5) and O(4) of the pyrimidine moiety hydrogen bond to the amide and the hydroxyl group of Thr37,
which is known to increase the redox potential of the FMN in OYE by withdrawing electrons from the ring, as indicated by studies with the Thr37Ala mutant [6]. Additionally, hydrogen bonds are formed between O(2) and Arg239 and Gln110 as well as between N(3) and Gln110 that is further fixed by a hydrogen bond to Glu185 underneath the FMN binding site. The amide group of Ala68 is brought into hydrogen bonding distance to O(4) by a bulge in the C␣ trace of strand 2 between residues 66 and 69 that locally disturbs the hydrogen bonding network of the  barrel. The residues that contact FMN by their side chains are strictly conserved among the OYE family. On the other hand, the remaining residues that form main chain contacts to the flavin vary between OPR1 and OYE and also within the OYE family. Neverthe-
Crystal Structure of OPR1 423
Figure 4. Substrate Binding Site of OPR1 (a) Stereo view of the substrate binding site of the OPDA-OPR1 structure (green) overlaid with the structure of OYE (beige) after structural alignment. Residues are numbered according to the OPR1 sequence. The FMN of OYE that superimposes perfectly with that of OPR1 is left out for clarity. The substrate electron density as it appeared in the 2Fo-Fc omit map of the final model is calculated at 2.0 A˚ and contoured at 1.0 . It shows the orientation of the substrate relative to the FMN and the general acid Tyr192. The figure illustrates the high structural conservation of residues near the cyclopentenone and the differing contributions of loop L6(OYE) and L3(OPR) to the active site. (b) Schematic diagram of the active site of the OPDA-OPR1 complex made up by the highly conserved catalytic site and the variable specificity site. Atoms C16-C18 of OPDA drawn as dotted lines are not ordered in the structure. Boxed residues are conserved in the OYE family, while the loops L5/L6 and L1-3 that form the specificity site vary widely in length and sequence.
less their C␣ positions, essential for the observed hydrogen bonds, are structurally well conserved, showing an rmsd of only 0.7 A˚ between OPR1 and OYE. Substrate Binding Site The substrate binding site is located above the si face of the flavin and contains a large tunnel that is built by residues of L6 and L2 and by L3, which forms a molecular clamp by stretching across the complete (␣)8 barrel and contacting Tyr358 situated in the ultimate turn following helix ␣8 (Figures 1b and 4a). Inside the tunnel, a narrow cavity branches off toward the center of the barrel, with the bottom of this cavity being formed by the flavin pyrimidine ring. Upon substrate binding, the cyclopentenone ring of 9R,13R-OPDA becomes deeply buried in this cavity, while the carboxyalkyl chain extends into the tunnel toward L2 and the short pentenyl chain protrudes toward the exterior in the direction of the ribityl chain. Except for the last three carbon atoms
of the pentenyl chain, the orientation and conformation of the substrate molecule can be unequivocally derived from the electron density. Inside the cavity, the substrate carbonyl oxygen is hydrogen bonded to His187 N⑀2 and His190 N␦1 with distances of 3.0 A˚ and 2.9 A˚, respectively. The cyclopentenone ring stacks nearly parallel above the flavin placing C10 (⫽C) and C11 (⫽C␣) close to flavin N(5) (distance 4.1 A˚) and the hydroxyl group of Tyr192 (distance 3.9 A˚), respectively (Figure 4). The OPDA alkyl chains make numerous van der Waals contacts to residues lining the tunnel, predominantly to the aromatic rings of Tyr246, Tyr78, and Tyr358. The carboxylate group is anchored by two hydrogen bonds to the amide and hydroxyl group of Ser143 that is located in L3 near the opening of the active site tunnel. As the Arg142Met-OPR1 mutant was used for crystallization (see Experimental Procedures) it is conceivable that the substrate carboxylate group additionally binds to Arg142 in the wild-type enzyme. The difference in bind-
Structure 424
Figure 5. Proposed Mechanism of Substrate Reduction by OPR1 The hydride is transferred from FMN N(5) to C(10) of the activated 9R,13R-OPDA, whereas Tyr192 protonates C(11) from the opposite side, shown here as a concerted reaction [7].
ing energy, however, does not seem to change the overall reactivity of the enzyme, since the steady-state kinetic properties of OPR1 and the stereoselectivity with respect to the cis OPDA enantiomers remained unaffected. In the PEG400-OPR1 complex, PEG400 adopts a conformation similar to the alkyl chains of OPDA, while the position of the cyclopentenone oxygen is occupied by a highly ordered water molecule that is bound to His187, His190, and PEG400 O17. The residues of the PEG400-OPR1 complex that form the active site cavity move only slightly compared to the OPDA-OPR1 structure. In the PEG400-free structure, the cavity is filled by four ordered water molecules. The only significant change in conformation of the surrounding residues is observed for Tyr246 that lost its partner for van der Waals contacts and turns away from the active site. This conformation of Tyr246 is stabilized by a hydrogen bond to a water molecule that also hydrogen bonds to Tyr78. As indicated by the rather small conformational changes in the three structures, the active site cavity seems to be rather rigid, like preshaped, which is further supported by the low average temperature factor of 18.5 A˚2 of the concerned residues. Two hydrogen bonds between Tyr192 OH and Tyr78 OH at the top of the cavity and between Asn247 O␦1 and Gln140 N⑀2 contribute to the observed rigidity. By contrast, residues of the substrate binding tunnel especially of the molecular clamp formed by L3 move according to the different sizes of OPDA and PEG400. Mechanism of Substrate Reduction In the OYE family, NADPH oxidation and substrate reduction is performed by a bi-bi ping pong mechanism with NADPH and the unsaturated substrate using the same binding site. The reduction of the substrate olefinic bond by OYE has been shown to proceed via hydride transfer from the flavin N(5) to the substrate C and protonation of the substrate C␣ from the opposite site by Tyr192 (Tyr196 in OYE). The function of Tyr196 as a general acid in OYE was demonstrated by mutagenesis of Tyr196 to phenylalanine, resulting in a nearly 106-fold decrease of the rate of oxidation with cyclohexenone
[7]. Most notably, using 1-nitrocyclohexene as the substrate, the Tyr196Phe mutant enzyme was still capable of hydride transfer but did not catalyze the subsequent protonation of nitronate to nitrocyclohexane anymore [7]. The OPDA-OPR1 structure—being the first structure of an enzyme-substrate complex in the OYE-family— confirms the mechanism of substrate reduction that has been proposed on the basis of detailed mechanistic studies with active site mutants of OYE and of the related estrogen binding protein (EBP1) from Candida albicans [5, 7, 31] (Figure 5). The substrate carbonyl oxygen that superimposes perfectly with the hydroxyl group of the ligand para-hydroxybenzaldehyde (PHB) in the PHBOYE structure [3] is hydrogen bonded to His187 and His190, leading to polarization of the olefinic bond and, thus, to activation of C for nucleophilic attack. In OYE and several other members of the family, His190 is replaced by an asparagine. However, the N␦1 of His190 in OPR1 occupies the same position as the corresponding Asn194 N␦1 in OYE. Therefore, the electronic effects on the substrate oxygen are likely to be quite similar. The C atom of the substrate is positioned at a distance of 4.1 A˚ to the flavin N(5) and forms an angle of 106⬚ with the N(5)-N(10) and of 73⬚ with the N(5)-C(4) atoms, which results in a shift of C toward the C(4) locus. While the distance is slightly larger than the typically observed value of ⵑ3.5 A˚, the angular geometry resembles the data of other substrate-flavoprotein complexes (reviewed in [32]), for example, of MurB and quinone reductase [33–35], that also catalyze hydrogenation reactions of ␣,-unsaturated substrates. It should be noted that these structures represent the nonreactive complexes between oxidized substrate and oxidized flavin. In contrast to MurB and quinone reductase, the substrate of OPR1 is neither involved in extensive hydrogen bonding nor stacked against an upper aromatic side chain and may thus avoid unfavorable close contacts to the oxidized flavin more easily. While in flavoproteins like D-amino-acid-oxidase or flavodoxin the flavin is essentially planar, in the oxidized and the reduced form [36– 38], the FMN is planar in oxidized OYE but shows a puckering of the N(5) locus in the reduced OYE structure that is associated with a butterfly bending of the isoalloxazine ring by 15⬚. If one superimposes the active sites
Crystal Structure of OPR1 425
of the OPDA-OPR1 complex and the reduced OYE structure to take into account this comparatively large bending of FMNH2 [39], the distance between the substrate C and N(5) of FMNH2 is 3.6 A˚, close to the expected value. The substrate binding site architecture also helps to explain the observed stereoselectivity. When the enantiomeric 9S,13S-OPDA that is not reduced by OPR1 is modeled into the active site in compliance with the mechanistic requirements (i.e., activation by hydrogen bonds to His187 and His190 and proper orientation of C for hydride transfer), sterically unfavorable contacts are formed between the substrate carbons C7, C8, and C14 and the FMN cofactor and Tyr358. Solvent exclusion is thought to be necessary for efficient enzymatic hydride transfer [32, 40]. Hence, the active site of many flavoenzymes is buried below the protein surface granting access only during substrate admission or product release. In OPR1, however, the catalytic site is made solvent inaccessible by substrate binding per se. OPDA exactly fits to the cavity above the flavin, as it is also seen for substrate binding of medium chain acyl-CoA dehydrogenase [41]. Thus, the isoalloxazine ring of the flavin becomes almost completely covered upon substrate binding. Furthermore, the molecular surface of the substrate atoms C5 to C14 that include the cyclopentenone ring is rendered inaccessible upon binding of OPDA so that the reaction can proceed without interference from solvent molecules. The protein tunnel that forms extensive van der Waals contacts to the substrate alkyl chains seems to be a unique feature of OPR1. It has no equivalent in OYE, whose active site cavity—lacking the participation of L3—opens much earlier (Figure 6). Consistently, modeling of an 9R,13R-OPDA-OYE complex suggests only weak binding of C6-C8 and leaves the rest of the carboxyalkyl chain protruding freely into the solvent (data not shown). In OPR1, anchoring of the substrate alkyl chains in the hydrophobic tunnel seems to support the correct positioning of the cyclopentenone ring inside the active site. This idea is confirmed by activity studies with OPR1 which show that OPR1 is about 20-fold more active with OPDA as substrate than with cyclopentenone (J.S. and A.S., unpublished data). Substrate Specificity of OPR1 and of the OYE Family A remarkable aspect of the OPR1 and OYE structures is the high structural conservation of catalytically important residues within the active site cavity and the pronounced structural differences in the loops adjacent to it. A closer inspection of sequence conservation in the OYE family reveals that all residues contributing to the cavity above the flavin pyrimidine ring in OPR1 are invariant, whereas the residues forming the hydrophobic tunnel—especially the residues comprising L3 and L6— show hardly any conservation. Thus, the substrate binding sites in the OYE family seem to be composed of a highly conserved catalytic site, the cyclopentenone binding cavity in OPR1, and a variable specificity site that, enclosing the two substrate alkyl chains in OPR1, is responsible for substrate recognition and binding (Figure 4b).
Figure 6. Surface Representation of the Active Site Viewed from Strand 2 (a) Structure of the OPDA-OPR1 complex. (b) Structure of the PHBOYE complex [3]. The molecular surface of the flavin is colored red. Structurally corresponding residues of OYE and OPR1 are depicted in the same colors (e.g., OPR1/Q39 and OYE/M39). Loop L3, crucial for the formation of the active site tunnel in OPR1, is located distant from the active site in OYE. Consequently, the active site cavity of OYE is much more open than in OPR and lined by residues that lie inside the tunnel of OPR1.
This observation and, moreover, the mode of substrate binding in the OPDA-OPR1 complex further support the assumption that ␣,-unsaturated carbonyl compounds are the physiological substrates of these enzymes. In this context, it is noteworthy that the speci-
Structure 426
ficity site of OYE does not support the binding of a hydrophobic alkyl chain as present in OPDA, which is also reflected by the lower activity of OYE with OPDA compared to OPR1 (F. Schaller, personal communication) and argues against an OPDA-like physiological substrate of OYE. Substrate specificity has turned out to be a crucial factor in the characterization of the OPR isozymes. OPR1, the first cloned OPR isozyme, was initially thought to be directly involved in the biosynthesis of JA [13]. However, in vitro studies with OPR1 from A. thaliana and L. esculentum demonstrated that it does not reduce the JA precursor 9S,13S-OPDA but the enantiomeric 9R,13R-OPDA[19], what is now confirmed by the observed active site geometry of OPR1. Recent activity studies with OPR3 [18], the induction of the opr3 gene by wounding [43], and the complementation of a malesterile phenotype of an opr3 knockout mutant by exogenous JA [44] strongly suggest that OPR3 is the isozyme responsible for JA biosynthesis. From the crystal structure of OPR1, several assumptions about the physiological function of OPR1 can be made. With regard to the active site geometry of OPR1 and the observed efficient turnover of (2E)-alkenals in vitro [42], aliphatic ␣,-unsaturated carbonyl compounds are potential substrates of OPR1 in vivo. These compounds are formed during the oxidative burst in wounded and diseased plant tissues [45, 46], where they act as cytotoxic agents but are also involved in the activation of defense-related genes. In view of the good fit of the OPDA ring to the active site of OPR1, however, 9R,13R-OPDA itself or a 9R,13ROPDA-like cyclopentenone could be a relevant substrate in vivo. Thus, the proposed function of OPR1 removing 9R,13R-OPDA in order to maintain a stereochemically homogenous pool of 9S,13S-OPDA for JA biosynthesis remains a possibility [19]. Alternatively, OPR1 could be involved in a pathway parallel to JAsynthesis, i.e., the biosynthesis or the metabolism of a biologically active cyclopentanone/-pentenone, for example 9,10-dihydrojasmonic acid or dinor-OPDA [47– 50]. Like JA and OPDA, these signaling molecules contribute to the specific “oxylipin signature” [48, 51, 52] of plants, which changes in response to abiotic or biotic stresses.
Biological Implications Being constantly exposed to numerous potential aggressors, plants have evolved complex mechanisms to protect themselves against herbivores and pathogens. Apart from its role in regulating various developmental processes, JA is a potent signal molecule in the defense system of plants, where it regulates the expression of many wound-activated and defense-related genes. It has recently been shown that compounds structurally related to JA or JA precursors (collectively called jasmonates or octadecanoids) are biologically active themselves in plant defense, for example, by activating alkaloid biosynthesis. OPR catalyzes a key step in JA biosynthesis, the reduction of 9S,13S-OPDA to OPC8:0. Moreover, the OPRs are members of the OYE family,
flavoproteins from bacteria, fungi, or plants that are able to reduce the olefinic bond of ␣,-unsaturated carbonyl compounds by oxidizing NADPH, a reaction rather uncommon in flavoprotein chemistry. Except for the OPRs, the physiological context of these enzymes is not known. We have solved the structure of OPR1 from tomato, that specifically reduces 9R,13R-OPDA [19], and the structures of OPR1 complexed with its substrate 9R,13ROPDA and with the polyether PEG400. The OPDA-OPR1 complex confirms the mechanism of substrate reduction proposed on the basis of biochemical studies with other members of the OYE family. Remarkably, all amino acid residues that contact the reacting substrate atoms are strictly conserved in the OYE family and show almost identical conformations in OPR1 and in OYE. Thus, it can be assumed that ␣,-unsaturated carbonyl compounds are the likely natural substrates of this family. The long hydrophobic tunnel in OPR1, allowing for specific binding of the substrate’s carboxyalkyl chain, as well as the overall active site architecture that explains the observed stereoselectivity strongly argue for a 9R,13ROPDA-like substrate of OPR1 in vivo. Thus, OPR1 is likely to be involved in the biosynthesis or the metabolism of signaling molecules that contribute to the oxylipin signature in plants. A similar role can be inferred for the remaining OPR isozymes that are not directly involved in the biosynthesis of JA itself. Experimental Procedures Expression and Purification of OPR1 The OPR1 open reading frame from L. esculentum was cloned into the StuI and KpnI restriction sites of the E. coli expression plasmid pGEX-G, and the protein was purified as an N-terminal fusion with glutathione S-transferase as described [42]. Incubation with the protease Factor Xa (Factor Xa cleavage and removal kit; Roche Diagnostics, Rotkreuz, Switzerland) resulted in partial degradation of OPR1. N-terminal sequence analysis of the degradation products identified the site of cleavage as the Arg142/Ser143 peptide bond of OPR1. Site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) was employed to substitute Arg142 with methionine. The mutant protein was expressed in E. coli and purified from bacterial extracts as described [42]. After Factor Xa digestion, a homogenous protein preparation was obtained.
Crystallization and Data Collection Crystals were grown at 18⬚C by the sitting drop vapor diffusion method in droplets composed of one part protein solution (10 mg/ ml, in 10 mM MOPS/KOH [pH 7.0]) and two parts reservoir solution (100 mM Tris/HCl [pH 7.5], 1.45 M (NH4)2SO4, 2% PEG400), reaching a final size of 200 ⫻ 60 ⫻ 30 m3. The triclinic crystals have unit cell parameters a ⫽ 53.4 A˚, b ⫽ 71.3 A˚, c ⫽ 71.5 A˚, ␣ ⫽ 63.7⬚,  ⫽ 83.9⬚, and ␥ ⫽ 77.4⬚, corresponding to two molecules per asymmetric unit. To obtain a PEG400-free structure, the crystals were soaked in 10 mM Tris/HCl [pH7.5], 1.55 M (NH4)2SO4 for 12 hr, changing the buffer four times during this time. To obtain the 9R,13R-OPDA-OPR1 complex, the crystals were subsequently soaked in the same buffer complemented with racemic cis-OPDA (Cayman Chem. Co., Ann Arbor, MI; final concentration 2.5 mM), since it was shown that the enantiomeric 9S,13S-OPDA is no substrate of OPR1 [53]. After freezing the crystals in cryoprotectant (10 mM Tris/HCl [pH7.5], 1.55 M (NH4)2SO4, 35% glycerol), diffraction data were collected at 100K at the Wiggler beamlines BW7a and BW6 at the Deutsche Elektronen Synchrotron (Hamburg, Germany). All data sets were indexed, integrated, and scaled with DENZO and SCALE-
Crystal Structure of OPR1 427
PACK [54]. Data collection and refinement statistics are summarized in Table 1. Attempts were undertaken to obtain a nicotinamid dinucleotideOPR1 complex, since no information about the NADPH binding mode and the structural origin of the preference of NADPH over NADH in OPR and OYE is available so far. As already reported for OYE, no significant binding of NADP⫹ to OPR1 was observed after soaking at different conditions. Replacing PEG400 with NADP⫹ in the crystallization buffer, however, yielded unregularly grown nondiffracting crystals, whereas no crystals grew in NADP⫹-free buffer. Further work is in progress to obtain the structure of a nicotinamid dinucleotide-OPR complex.
Structure Solution and Refinement The PEG400-OPR1 structure was solved by molecular replacement performed with the program AMoRe [55]. The search model was generated from the coordinates of OYE by resetting the B factors to 20 A˚, replacing nonconserved residues by alanine, and eliminating residues in the loop regions of OYE that are missing in the aligned OPR1 sequence. After the rotational search at 3.5 A˚ resolution, two solutions were obtained, accounting for the two molecules in the asymmetric unit, that resulted in a correlation coefficient of 30.4% and an R value of 47.9%. Initial phases of the PEG400-free and of the OPDA complexed structure were calculated by using the coordinates of the refined PEG400-OPR1 structure as a search model. After initial rigid body minimization, refinement was performed by alternating model building carried out with the program O [56] and crystallographic refinement using CNS [57]. The refinement procedure included simulated annealing, positional refinement, and restrained temperature factor refinement using the parameters of Engh and Huber [58] and maximum likelihood algorithms as provided by CNS. Finally, water molecules were inserted automatically and checked manually by inspection of the Fo-Fc map. For all three models, noncrystallographic symmetry (ncs) restraints were applied, except for the last cycle of refinement. The flavin cofactor and the ligands were not included in the model during the first cycles of refinement; thereafter, they could be easily built into the clearly defined electron density. After releasing the ncs restraints, minor conformational differences of surface residues that form contacts to neighboring protomers became visible. Excluding these residues, both molecules of the asymmetric unit are highly similar, exhibiting an rmsd of 0.2 A˚ after structural alignment. Due to disorder, residues 283–288 and the N-terminal residues 1–10 remained undetermined in the electron density map. Removing PEG400 resulted in a higher flexibility of the PEG400-free structures, as reflected by six further residues being disordered and the higher overall temperature factors of the OPR1 and the 9R,13R-OPDAOPR1 structures (Table 1). Searches for structural neighbors were performed using the program TOP [59]. Sequence comparisons of the OYE family were made using GCG [60] and include the following family members (sequence identity with respect to OPR1, L. esculentum, is given in parenthesis): OPR3 (A. thaliana, 54%) [18], GTN reductase (47%) [14], morphinone reductase (44%) [10], NEM reductase (43%) [12], PETN reductase (43%) [9], XenB reductase (41%) [8], OYE1 (40%) [29], estrogen binding protein (38%) [11]. Figures were drawn with MOLSCRIPT [61], Dino [62], Setor [63], GRASP [64] and Raster3D [65].
Acknowledgments We would like to thank Gleb Bourenkow, Hans-Dieter Bartunik (MPG), and Ehmke Pohl (EMBL) of the Deutsche Elektronen Synchrotron, Hamburg, for assistance with data collection. This work was supported by research grants from the Swiss National Science Foundation to A.S. (31-46818.96) and to P.M. and A.S. (31-59047.99).
Received: January 22, 2001 Revised: April 6, 2001 Accepted: April 9, 2001
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