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 Km = 153 and 459 μm, respectively) and higher catalytic efficiencies (kcat/Km = 0.0179 and 0.0444 μm–1 min–1, respectively) with these acyl substrates compared with benzoate (apparent Km = 867 μm, kcat/Km = 0.0046 μm–1 min–1). Notably, SA specifically and reversibly inhibits PBS3 activity with an IC50 of 15 μm. 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
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