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

Okrent RA, Brooks MD, Wildermuth MC - J. Biol. Chem. (2009)

Bottom Line: In gh3.12/pbs3 mutants of Arabidopsis thaliana, induced total SA accumulation is significantly compromised as is SA-dependent gene expression and plant defense.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.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)).

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA.

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.

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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.
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fig3: 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.

Mentions: PBS3 Activity Is Inhibited by SA but Not by Other Phytohormones—As PBS3 impacts SA metabolism and the most active acyl substrates of PBS3 are structurally similar to SA, we then sought to determine whether SA could inhibit PBS3 activity. To test this, we assayed the effect of increasing concentrations of SA on PBS3-catalyzed pABA-Glu formation. As shown in Fig. 3, SA dramatically reduced the amino acid conjugation activity of PBS3 with an estimated IC50 of 15 μm. To determine whether the inhibition of PBS3 by SA was competitive, we added excess (saturating) pABA to the reaction mixture with 15 μm SA (the IC50). Full activity with maximal velocity was restored (data not shown). This suggests that SA acts as a competitive inhibitor of PBS3 activity.


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

Okrent RA, Brooks MD, Wildermuth MC - J. Biol. Chem. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665095&req=5

fig3: 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.
Mentions: PBS3 Activity Is Inhibited by SA but Not by Other Phytohormones—As PBS3 impacts SA metabolism and the most active acyl substrates of PBS3 are structurally similar to SA, we then sought to determine whether SA could inhibit PBS3 activity. To test this, we assayed the effect of increasing concentrations of SA on PBS3-catalyzed pABA-Glu formation. As shown in Fig. 3, SA dramatically reduced the amino acid conjugation activity of PBS3 with an estimated IC50 of 15 μm. To determine whether the inhibition of PBS3 by SA was competitive, we added excess (saturating) pABA to the reaction mixture with 15 μm SA (the IC50). Full activity with maximal velocity was restored (data not shown). This suggests that SA acts as a competitive inhibitor of PBS3 activity.

Bottom Line: In gh3.12/pbs3 mutants of Arabidopsis thaliana, induced total SA accumulation is significantly compromised as is SA-dependent gene expression and plant defense.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.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)).

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA.

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.

Show MeSH
Related in: MedlinePlus