PDBsum entry 3hgo

Oxidoreductase

PDB id: 3hgo

Contents

Protein chains

367 a.a. *

Ligands

FMN ×2

Waters ×501

* Residue conservation analysis

PDB id:

3hgo

Name:

Oxidoreductase

Title:

Crystal structure of the f74y/h244y opr3 double mutant from tomato

Structure:

12-oxophytodienoate reductase 3. Chain: a, b. Synonym: 12-oxophytodienoate-10,11-reductase 3, opda-reductase 3, leopr3. Engineered: yes. Mutation: yes

Source:

Solanum lycopersicum. Tomato. Organism_taxid: 4081. Gene: opr3. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.30Å R-factor: 0.200 R-free: 0.259

Authors:

T.Clausen,C.Breithaupt

Key ref:

C.Breithaupt et al. (2009). Structural basis of substrate specificity of plant 12-oxophytodienoate reductases. J Mol Biol, 392, 1266-1277. PubMed id: 19660473 DOI: 10.1016/j.jmb.2009.07.087

Date:

14-May-09 Release date: 25-Aug-09

Protein chains

Q9FEW9  (OPR3_SOLLC) -  12-oxophytodienoate reductase 3 from Solanum lycopersicum

Seq: 396 a.a.

Struc: 367 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.3.1.42 - 12-oxophytodienoate reductase.
• Reaction: (1S,2S)-OPC-8 + NADP⁺ = (9S,13S,15Z)-12-oxophyto-10,15-dienoate + NADPH + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1016/j.jmb.2009.07.087

J Mol Biol 392:1266-1277 (2009)

PubMed id: 19660473

Structural basis of substrate specificity of plant 12-oxophytodienoate reductases.

C.Breithaupt, R.Kurzbauer, F.Schaller, A.Stintzi, A.Schaller, R.Huber, P.Macheroux, T.Clausen.

ABSTRACT

12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate [(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate. To uncover the molecular determinants of this remarkable enantioselectivity, we determined the crystal structures of OPR1 and OPR3 in complex with the ligand p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex and further biochemical and mutational analyses revealed that two active-site residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical for substrate filtering. The relatively smaller OPR3 residues allow formation of a wider substrate binding pocket that is less enantio-restrictive. Substitution of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3 mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence analysis of the OPR family supports the filtering function of Tyr78 and Tyr246 and allows predictions with respect to substrate specificity and biological function of thus far uncharacterized OPR isozymes. The discovered structural features may also be relevant for other stereoselective proteins and guide the rational design of stereospecific enzymes for biotechnological applications.

Selected figure(s)

Figure 2.
Fig. 2. Binding of PHB to OPR1 and OPR3. (a) 2F[o ]− F[c] omit electron density map of the complex structures of OPR1:PHB (left) and OPR3:PHB (right), at 2.30- and 2.07-Å resolution, contoured at 1.0σ. For map calculation, PHB was omitted from the model. (b) Stereo view of the superposition of the active-site cavities of OPR1:PHB (light blue) and OPR1:(9R,13R)-OPDA (green). The PHB ligand is shown in dark blue and OPDA in yellow. In addition, the ribbon structure of OPR1 is shown. (c) Stereo view of the superposition of the active-site cavities of OPR1:PHB (light blue), OPR3:PHB (yellow), and the OPR3 double mutant OPR3YY (green). The PHB ligand is shown in dark blue (OPR1) and orange (OPR3). In addition, the ribbon structure of OPR3 is shown. Residues are numbered according to the OPR3 sequence.

Figure 3.
Fig. 3. Substrate binding to OPR1 and OPR3. (a) Stereo view of the active-site cavity of the OPR1:(9R,13R)-OPDA complex. In addition to the molecular surfaces of OPR1 (grey; surface of FMN: green) and of the substrate (9R,13R)-OPDA (yellow), Tyr246 and Tyr78 that narrow the opening of the cavity as well as (9R,13R)-OPDA are shown as ball-and-stick models (blue). (b) Stereo view of the active-site cavity of a modeled OPR3:(9R,13R)-OPDA complex. Surfaces were colored as in (a). The complex was obtained by transferring the substrate's coordinates of the aligned OPR1:(9R,13R)-OPDA complex to the OPR3 structure. Protein residues of OPR3 and the substrate's carboxy alkyl chain (shortened in the figure for clarity) clash in the model because OPR3 lacks the tunnel that accommodates the carboxy alkyl chain in OPR1. In OPR3, these clashes can be easily avoided by a change in the conformation of atoms C1 to C8 of the carboxy alkyl chain. In comparison to OPR1, the opening of the OPR3 cavity is lined by His244 and Phe74, resulting in a wider entrance and leaving more space near the stereo centers of the substrate.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 392, 1266-1277) copyright 2009.

Figures were selected by an automated process.