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Arabidopsis GH3.12 (PBS3) conjugates amino acids to 4-substituted benzoates and is inhibited by salicylate.
J Biol Chem. 2009 Apr 10; 284(15):9742-54.JB

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.

Authors+Show Affiliations

Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19189963

Citation

Okrent, Rachel A., et al. "Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-substituted Benzoates and Is Inhibited By Salicylate." The Journal of Biological Chemistry, vol. 284, no. 15, 2009, pp. 9742-54.
Okrent RA, Brooks MD, Wildermuth MC. Arabidopsis GH3.12 (PBS3) conjugates amino acids to 4-substituted benzoates and is inhibited by salicylate. J Biol Chem. 2009;284(15):9742-54.
Okrent, R. A., Brooks, M. D., & Wildermuth, M. C. (2009). Arabidopsis GH3.12 (PBS3) conjugates amino acids to 4-substituted benzoates and is inhibited by salicylate. The Journal of Biological Chemistry, 284(15), 9742-54. https://doi.org/10.1074/jbc.M806662200
Okrent RA, Brooks MD, Wildermuth MC. Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-substituted Benzoates and Is Inhibited By Salicylate. J Biol Chem. 2009 Apr 10;284(15):9742-54. PubMed PMID: 19189963.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Arabidopsis GH3.12 (PBS3) conjugates amino acids to 4-substituted benzoates and is inhibited by salicylate. AU - Okrent,Rachel A, AU - Brooks,Matthew D, AU - Wildermuth,Mary C, Y1 - 2009/02/02/ PY - 2009/2/5/entrez PY - 2009/2/5/pubmed PY - 2009/5/21/medline SP - 9742 EP - 54 JF - The Journal of biological chemistry JO - J Biol Chem VL - 284 IS - 15 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/19189963/Arabidopsis_GH3_12__PBS3__conjugates_amino_acids_to_4_substituted_benzoates_and_is_inhibited_by_salicylate_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(20)32234-1 DB - PRIME DP - Unbound Medicine ER -