X-ray structure of Arabidopsis At1g77680, 12-oxophytodienoate reductase isoform 1



In plants, octadecanoid-mediated signaling pathways provide defense from pathogen attack, response to physical damage, stimulation of male fertility, and a mechanism for interspecies communication.1–3 In the jasmonic acid branch of these biosynthetic pathways, reduction of 9S, 13S-OPDA is a key stereospecific reaction. The central role of 9S, 13S-OPDA, which has structural similarity to mammalian prostaglandins, lends significance to characterization of the enzyme(s) responsible for its reduction. This enzyme is an NAD(P)H-dependent flavoprotein oxidoreductase named 12-oxophytodienoate reductase (OPR, EC Although five OPR isoforms have been identified in Arabidopsis by genome analysis, only OPR3 is presently known to reduce the physiologically relevant 9S, 13S-OPDA.4 In contrast, OPR1 and OPR2 give exclusive reduction of the 9R, 13R- stereoisomer of OPDA, while OPR3 can also reduce this alternative diastereomer. The biological role of the alternative diastereomers of OPDA is not known. Furthermore, the functions of OPR4 and OPR5 are not known, although both isoforms are known to be expressed in certain tissues.

This work reports the 2.0-Å X-ray structure of Arabidopsis OPR1, identified to be encoded by At2g76680.5 Comparison of the Arabidopsis OPR1 structure with the Arabidopsis OPR3 (1Q45) and tomato OPR1 (1ICQ6) structures revealed a third backbone conformation at the putative substrate-binding loop in the newest structure. The variability in configuration observed at this critical region of the protein structure further emphasizes the need for detailed structural analysis to understand the role of each OPR isoform in the octadecanoid-dependent signaling pathways.


OPDA, 12-oxo-10,15(Z)-phytodienoic acid; OPR, 12-oxophytodienoate reductase; RMS, root mean square.

Materials and Methods.

The cDNA preparation used for cloning was obtained by conversion of the total mRNA isolated from an Arabidopsis tissue culture using reverse transcriptase.7 A two-step PCR and Gateway method (Invitrogen, Carlsbad, CA) was used for cloning from the cDNA preparation. The identity of the cloned OPR1 gene was verified by DNA sequencing. The vector pQE80 (Qiagen, Seattle, WA) was altered to support the Gateway method and to give an N-terminal fusion of S-tag-(His)6-maltose binding protein to OPR1. Escherichia coli Rosetta (DE3) pLacI Rare cells transformed with the OPR1 expression plasmid were grown in terrific broth medium in 2-L beverage bottles and induced by IPTG.8 The OPR1 fusion was purified using Ni-IDA chromatography and proteolyzed in ∼ 95% yield using tobacco etch virus protease. Further details of the cloning, expression, and purification will be reported elsewhere.

OPR1 (10 mg/ml in 10 mM Tris, pH 8.0, 100 mM NaCl, 0.3 mM TCEP) was crystallized by the vapor diffusion/hanging drop method. The hanging drops consisted of 2 μL of protein solution plus 2 μL of well solution (8.75% to 10% mmePEG 5000, 0.1 M triethanolamine, pH 8.0, 0.275 M to 0.35 M glycine). Crystals were harvested into a cryoprotectant solution (12% mmePEG 5000, 0.1 M triethanolamine, pH 8.0, 0.4 M glycine and 25% PEG 400) and flash cooled in a stream of nitrogen at 100°K. Diffraction data were collected at the Advanced Photon Source beamline 14-ID-B using a marCCD (165 mm) area detector and processed using HKL2000.9

Phases were solved by molecular replacement with SOMORE10 using PDB entry 1Q45 as a search model. The structure was refined using REFMAC11 from the CCP4 package12 as implemented in the CCP4i interface.13 Model building was performed using XFIT.14

Results and Discussion.

This structure was solved as part of the continuing efforts of the University of Wisconsin Center for Eukaryotic Structural Genomics on eukaryotic proteins (http://www.uwstructuralgenomics.org).

Table 1 shows the structure parameters obtained for OPR1. The protein is a monomer in solution based on preliminary biochemical evaluations. OPR1 is present as a monomer in the asymmetric unit, with one bound FMN and 113 ordered solvent molecules in the final model. The final R-factor and Rfree were 0.206 and 0.293 for the 20,780 unique reflections observed between 43.85 and 2.003 Å (96.9% complete). The RMS deviation of bond lengths and bond angles from the ideal values were 0.024 Å and 1.89°, respectively. Coordinates for the crystal structure have been deposited in the PDB with accession number 1VJI.

Table I. Summary of Data Collection, Crystal Structure, and Refinement Statistics
  • a

    Numbers in parentheses indicate the highest resolution shell of 10.

  • b

    Rsym = Σ|I − 〈I〉|/Σ I, where I = observed intensity and 〈I〉 = average intensity obtained from multiple measurements.

  • c

    R-factor = Σ||F0| − |F0||/Σ|F0|, where |F0| = observed structure factor amplitude and |Fc| = calculated structure factor amplitude.

  • d

    Rfree, R-factor based on 5% of the data excluded from refinement.

Space groupC2221
Wavelength (Å)0.9640
Unit cell dimensionsa = 46.69, b = 88.07, c = 149.33
Resolution range (Å)a43.85 − 2.00 (2.05 − 2.00)
Unique reflections20780
Completeness (%)98.2 (92.0)
Redundancy3.6 (3.3)
Rsym (%)b6.50 (81.6)
R-factor (%)c20.6 (27.2)
Rfree (%)d29.3 (34.7)
Average B factor (Å2)31.11
RMSD bond lengths (Å)0.024
RMSD bond angles (°)1.89

Figure 1(A) shows that OPR1 is a member of an α/β barrel fold family of FMN-containing oxidoreductases. This family was originally defined by structural studies of yeast Old Yellow Enzyme.15 The backbone atoms of Arabidopsis OPR1 and OPR3 overlay with an RMS deviation of 0.637 Å, whereas the backbone atoms of Arabidopsis OPR1 and tomato OPR1 (1ICQ) overlay with an RMS deviation of 0.513 Å. Residues found in Arabidopsis OPR1 that are within 5 Å of FMN include A30, P31, L32, T33, R34, E63, A64, Q106, E181, H183, N186, R235, I272, A303, G304, G305, F306, A324, Y325, G326, R327, W328, L330, F353, and Y354. A number of these residues are conserved in this family, and presumably form the enzyme active site. No electron density was observed from before S9 in the N-terminal, after E369 in the C-terminal, and from residues that form a loop between M276 and H287. The comparable loop in all OPR structures determined to date has likewise been disordered.

Figure 1.

A: Stereo representation of the X-ray structure of Arabidopsis OPR1 (1VJI) showing the position of FMN and the putative substrate-binding loop (violet). B: comparison of the positions of the putative substrate-binding loop of OPR1 (violet) with those observed in structures of Lycopersicon esculentum (tomato) OPR1 (grey) and Arabidopsis OPR3 (green). Positions obtained from alignment of the backbone atoms of each structure with Arabidopsis OPR1. The position of bound 9R,13R-OPDA in the 1ICQ structure is also shown (grey).The Arabidopsis OPR1 loop consists of P132-P149, the tomato OPR1 loop consists of residues G136-R153, and the Arabidopsis OPR3 loop consists of I133-Y150. In the tomato OPR1 loop, the backbone traces from 1ICQ (with 9R, 13R-OPDA bound) and 1ICS (with PEG bound) are indistinguishable.

We previously reported that the only major difference in the position of backbone atoms of Arabidopsis OPR3 and tomato OPR1 was in the position and identity of residues comparable to the Lβ3 substrate-binding loop identified in tomato OPR1. In Figure 1(B), the Lβ3 loop found in three different tomato OPR1 structures (1ICQ, 1ICP, 1ICS) are shown as a grey color (residues G136–R153), whereas the comparable Arabidopsis OPR1 loop is shown as a violet color (residues P132–P149), and the Arabidopsis OPR3 loop is shown as a green color (residues I133–Y150). Among these five structures from closely related enzymes, including isoforms from the same species, there are substantial differences in the position of this putative substrate-binding loop. Further studies are clearly required to eludicate the role of this variable loop in the reactions possible with this catalytically versatile family.


We acknowledge support from NIGMS GM645980 and BioCARS at the Advanced Photon Source/Argonne National Laboratory. T.E.M. and P.G.B. are trainees of the NIH Institutional Biotechnology Pre-Doctoral Training Grant T32 GM08349.