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Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase.
J Biol Chem. 2010 Sep 24; 285(39):29780-6.JB

Abstract

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg(2+) or Mn(2+) for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4-9-fold reductions in k(cat)/K(m) relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.

Authors+Show Affiliations

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

20639576

Citation

Chen, Qingfeng, et al. "Kinetic Basis for the Conjugation of Auxin By a GH3 Family Indole-acetic Acid-amido Synthetase." The Journal of Biological Chemistry, vol. 285, no. 39, 2010, pp. 29780-6.
Chen Q, Westfall CS, Hicks LM, et al. Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase. J Biol Chem. 2010;285(39):29780-6.
Chen, Q., Westfall, C. S., Hicks, L. M., Wang, S., & Jez, J. M. (2010). Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase. The Journal of Biological Chemistry, 285(39), 29780-6. https://doi.org/10.1074/jbc.M110.146431
Chen Q, et al. Kinetic Basis for the Conjugation of Auxin By a GH3 Family Indole-acetic Acid-amido Synthetase. J Biol Chem. 2010 Sep 24;285(39):29780-6. PubMed PMID: 20639576.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase. AU - Chen,Qingfeng, AU - Westfall,Corey S, AU - Hicks,Leslie M, AU - Wang,Shiping, AU - Jez,Joseph M, Y1 - 2010/07/18/ PY - 2010/7/20/entrez PY - 2010/7/20/pubmed PY - 2010/10/22/medline SP - 29780 EP - 6 JF - The Journal of biological chemistry JO - J Biol Chem VL - 285 IS - 39 N2 - The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg(2+) or Mn(2+) for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4-9-fold reductions in k(cat)/K(m) relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/20639576/Kinetic_basis_for_the_conjugation_of_auxin_by_a_GH3_family_indole_acetic_acid_amido_synthetase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)89012-9 DB - PRIME DP - Unbound Medicine ER -