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Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate

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...

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Autores principales: Okrent, Rachel A., Brooks, Matthew D., Wildermuth, Mary C.
Formato: Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665095/
https://www.ncbi.nlm.nih.gov/pubmed/19189963
http://dx.doi.org/10.1074/jbc.M806662200
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author Okrent, Rachel A.
Brooks, Matthew D.
Wildermuth, Mary C.
author_facet Okrent, Rachel A.
Brooks, Matthew D.
Wildermuth, Mary C.
author_sort Okrent, Rachel A.
collection PubMed
description 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 μm, respectively) and higher catalytic efficiencies (k(cat)/K(m) = 0.0179 and 0.0444 μm(–1) min(–1), respectively) with these acyl substrates compared with benzoate (apparent K(m) = 867 μm, k(cat)/K(m) = 0.0046 μm(–1) min(–1)). Notably, SA specifically and reversibly inhibits PBS3 activity with an IC(50) 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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spelling pubmed-26650952009-04-20 Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate Okrent, Rachel A. Brooks, Matthew D. Wildermuth, Mary C. J Biol Chem Enzyme Catalysis and Regulation 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 μm, respectively) and higher catalytic efficiencies (k(cat)/K(m) = 0.0179 and 0.0444 μm(–1) min(–1), respectively) with these acyl substrates compared with benzoate (apparent K(m) = 867 μm, k(cat)/K(m) = 0.0046 μm(–1) min(–1)). Notably, SA specifically and reversibly inhibits PBS3 activity with an IC(50) 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. American Society for Biochemistry and Molecular Biology 2009-04-10 /pmc/articles/PMC2665095/ /pubmed/19189963 http://dx.doi.org/10.1074/jbc.M806662200 Text en Copyright © 2009, The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Enzyme Catalysis and Regulation
Okrent, Rachel A.
Brooks, Matthew D.
Wildermuth, Mary C.
Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate
title Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate
title_full Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate
title_fullStr Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate
title_full_unstemmed Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate
title_short Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate
title_sort arabidopsis gh3.12 (pbs3) conjugates amino acids to 4-substituted benzoates and is inhibited by salicylate
topic Enzyme Catalysis and Regulation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665095/
https://www.ncbi.nlm.nih.gov/pubmed/19189963
http://dx.doi.org/10.1074/jbc.M806662200
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