Arabidopsis GH3.12 (PBS3) conjugates amino acids to 4-substituted benzoates and is inhibited by salicylate

Abstract

Salicylate (SA, 2-hydroxybenzoate) is a phytohormone best known for its role as a critical mediator of local and systemic plant defense responses. In response to pathogens such as Pseudomonas syringae, SA is synthesized and activates widespread gene expression. In gh3.12/pbs3 mutants of Arabidopsis thaliana, induced total SA accumulation is significantly compromised as is SA-dependent gene expression and plant defense. AtGH3 subfamily I and II members have been shown to conjugate phytohormone acyl substrates to amino acids in vitro, with this role supported by in planta analyses. Here we sought to determine the in vitro biochemical activity and kinetic properties of GH3.12/avrPphB susceptible 3 (PBS3), a member of the uncharacterized AtGH3 subfamily III. Using a novel high throughput adenylation assay, we characterized the acyl substrate preference of PBS3. We found PBS3 favors 4-substituted benzoates such as 4-aminobenzoate and 4-hydroxybenzoate, with moderate activity on benzoate and no observed activity with 2-substituted benzoates. Similar to known GH3 enzymes, PBS3 catalyzes the conjugation of specific amino acids (e.g. Glu) to its preferred acyl substrates. Kinetic analyses indicate 4-aminobenzoate and 4-hydroxybenzoate are preferred acyl substrates as PBS3 exhibits both higher affinities (apparent K(m) = 153 and 459 microm, respectively) and higher catalytic efficiencies (k(cat)/K(m) = 0.0179 and 0.0444 microm(-1) min(-1), respectively) with these acyl substrates compared with benzoate (apparent K(m) = 867 microm, k(cat)/K(m) = 0.0046 microm(-1) min(-1)). Notably, SA specifically and reversibly inhibits PBS3 activity with an IC(50) of 15 microm. This suggests a general mechanism for the rapid, reversible regulation of GH3 activity and small molecule cross-talk. For PBS3, this may allow for coordination of flux through diverse chorismate-derived pathways.

FIGURE 1.

Reaction catalyzed by GH3 enzymes, shown with benzoate as the acyl substrate. Adenylation of the acyl substrate is followed by formation of the amino acid conjugate.

FIGURE 2.

PBS3 catalyzes the formation of amino acid conjugates with preferred acyl substrates. A, TLC analysis of 4-HBA amino acid conjugate formation by PBS3. B, HPLC analysis of 4-HBA-Glu formation by PBS3. Inset, verification of putative 4-HBA-Glu peak by LC-MS. Shown is Q-TOF MS/MS on m/z = 268.1 with 4-HBA-Glu structure. Likely fission site is indicated. C, TLC analysis of pABA-Glu formation by PBS3 as follows: pABA standard (left), reaction mixture (middle), and pABA-Glu standard (right). D, TLC analysis of the PBS3 reactions with vanillic acid (VA), 4-HBA, or trans-cinnamic acid (t-CA) as the acyl substrate and Glu. The arrow indicates a faint spot corresponding to trans-cinnamic acid-Glu. E, TLC of the PBS3 reaction with 4-HBA and Glu derivatives. AAD, l-2-aminoadipic acid; Gla, γ-carboxyl l-glutamic acid; MeGlu, l-gutamic acid γ-methyl ester. Images of TLC plates were converted to grayscale and adjustments of “levels” in Photoshop were performed. Independent experiments with different batches of purified, recombinant PBS3 produced similar results.

FIGURE 3.

Inhibition of PBS3 activity by SA. SA inhibits the formation of pABA-Glu catalyzed by PBS3. The reaction velocities were determined by monitoring the formation of pABA-Glu by HPLC of PBS3 reactions with 150 μm pABA and 10 mm Glu. Independent experiments yielded similar results.