Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

Abstract

12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.flavoprotein ͉ jasmonic acid biosynthesis ͉ plant defense ͉ oxylipin signature ͉ 12-oxophytodienoic acid F lavoproteins catalyze a wide variety of essential biochemical reactions, including electron transfer, dehydrogenation, and hydroxylation reactions. Old yellow enzyme (OYE), the first flavin-dependent enzyme identified (1), and homologues of OYE from bacteria, yeast, and plants are able to reduce the CAC double bond of ␣,␤-unsaturated carbonyl compounds, an activity that is rather uncommon for flavoenzymes (2, 3). This reaction has been shown to proceed by a ping-pong bi-bi mechanism, during which the FMN cofactor is reduced by NAD(P)H before the substrate is bound and reduced by hydride transfer to the substrate's C ␤ (4). Despite extensive efforts, the physiological substrate has been revealed only for one member of the OYE family, the plant enzyme 12-oxophytodienoate reductase (OPR) 3, which catalyzes one step in the biosynthesis of the plant hormone jasmonic acid (JA) (5, 6). JA and JA derivatives act as signaling compounds in the defense response against herbivores and pathogens and are involved in the regulation of various developmental processes, such as fruit ripening, pollen maturation, and senescence (7,8). JA is synthesized from ␣-linolenic acid, which is oxidized and cyclized, resulting in the cyclopentenone 9S,13S-12-oxophytodienoate (9S,13S-OPDA). OPR3 reduces 9S,13S-OPDA to the corresponding cyclopentanone, which is converted to JA by repeated ␤-oxidation ( Fig. 1) (9). Several OPR isozymes have been identified in plants, including 3 isoforms in Lycopersicon esculentum, 5 in Arabidopsis thaliana, and 13 in Oryza sativa (10, 11). O...

Empoasca leafhoppers attack wild tobacco plants in a jasmonate-dependent manner and identify jasmonate mutants in natural populations

“…This molecule is the substrate for allene oxide synthase (AOS) that forms (9Z, 13S, 15Z)-12, 13-epoxy-18:3, which subsequently is cyclized to an isomeric mixture of 12-oxo-phytodienoic acid (OPDA) by allene oxide cyclase (AOC) (31). OPDA is transported into the peroxisome where the C 10 -C 11 double bond in (9S, 13S)-OPDA is reduced by an OPDA reductase (OPR) (32). The reduced OPDA (OPC-8:0) undergoes three cycles of β-oxidation involving acyl-CoA transferase (ACX) (33) and finally forming (3R, 7S)-JA.…”

Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacter oxydans

“…S2). Similar to other OYE structures, [33][34][35] the overall structure of Gox0502 resembles the traditional hydrolase structure, which consists of a single domain fold comprising an eight-stranded parallel α/β barrel with approximate dimensions of 45 Å × 45 Å × 40 Å (Fig. 1).…”

“…Similar to other OYE proteins, [33][34][35] Gox0502 folds into an eightstranded parallel α/β barrel with approximate dimensions 45 Å × 45 Å × 40 Å. Structural superimposition of Gox0502 in free form with OYE proteins in complex with FMN and substrate as well as the structural model of Gox2684 revealed residues involved in catalysis (His172, Asn175, and Tyr177), substrate selection (Trp66 and Trp100), and product stereoselectivity determination (Trp66 and Trp100), respectively.…”

Structure of anAspergillus fumigatusold yellow enzyme (EasA) involved in ergot alkaloid biosynthesis

“…in the crystallization by a solvent-derived sulfate (Breithaupt et al, 2006). Most likely, the oligomerization observed in the EasA crystals is a crystal-packing artifact.…”

“…The OYE homolog 12-oxophytodienoate reductase 3 (OPR3) crystallizes with loop 6 inserted into the active site of the opposing monomer, making an inactivated homodimer (Breithaupt et al, 2006). OPR3 was likewise shown by size-exclusion chromatography, dynamic light scattering and analytical ultracentrifugation to exist in a monomer-dimer equilibrium in solution and was proposed to be regulated by homodimerization upon a phosphorylation event that was mimicked EasA active site.…”