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Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea.
Plant Physiol Biochem. 2016 Oct; 107:9-20.PP

Abstract

Phytohormone conjugation is one of the mechanisms that maintains a proper hormonal homeostasis and that is necessary for the realization of physiological responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases convert indole-3-acetic acid (IAA) to IAA-amino acid conjugates by ATP-dependent reactions. IAA-aspartate (IAA-Asp) exists as a predominant amide conjugate of auxin in pea tissues and acts as an intermediate during IAA catabolism. Here we report a novel recombinant indole-3-acetic acid-amido synthetase in Pisum sativum. In silico analysis shows that amino acid sequence of PsGH3 has the highest homology to Medicago truncatula GH3.3. The recombinant His-tag-PsGH3 fusion protein has been obtained in E. coli cells and is a soluble monomeric polypeptide with molecular mass of 69.18 kDa. The PsGH3 was purified using Ni(2+)-affinity chromatography and native PAGE. Kinetic analysis indicates that the enzyme strongly prefers IAA and L-aspartate as substrates for conjugation revealing Km(ATP) = 0.49 mM, Km(L-Asp) = 2.2 mM, and Km(IAA) = 0.28 mM. Diadenosine pentaphosphate (Ap5A) competes with ATP for catalytic site and diminishes the PsGH3 affinity toward ATP approximately 1.11-fold indicating Ki = 8.5 μM. L-Tryptophan acts as an inhibitor of IAA-amido synthesizing activity by competition with L-aspartate. Inorganic pyrophosphatase (PPase) hydrolyzing pyrophosphate to two phosphate ions, potentiates IAA-Asp synthetase activity of PsGH3. Our results demonstrate that PsGH3 is a novel enzyme that is involved in auxin metabolism in pea seeds.

Authors+Show Affiliations

Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland. Electronic address: maciejost@umk.pl.Department of Genetics, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.Department of Genetics, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27235647

Citation

Ostrowski, Maciej, et al. "Cloning and Biochemical Characterization of Indole-3-acetic Acid-amino Acid Synthetase PsGH3 From Pea." Plant Physiology and Biochemistry : PPB, vol. 107, 2016, pp. 9-20.
Ostrowski M, Mierek-Adamska A, Porowińska D, et al. Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea. Plant Physiol Biochem. 2016;107:9-20.
Ostrowski, M., Mierek-Adamska, A., Porowińska, D., Goc, A., & Jakubowska, A. (2016). Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea. Plant Physiology and Biochemistry : PPB, 107, 9-20. https://doi.org/10.1016/j.plaphy.2016.05.031
Ostrowski M, et al. Cloning and Biochemical Characterization of Indole-3-acetic Acid-amino Acid Synthetase PsGH3 From Pea. Plant Physiol Biochem. 2016;107:9-20. PubMed PMID: 27235647.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea. AU - Ostrowski,Maciej, AU - Mierek-Adamska,Agnieszka, AU - Porowińska,Dorota, AU - Goc,Anna, AU - Jakubowska,Anna, Y1 - 2016/05/20/ PY - 2016/04/25/received PY - 2016/05/18/revised PY - 2016/05/18/accepted PY - 2016/5/29/entrez PY - 2016/5/29/pubmed PY - 2017/3/28/medline KW - Auxin conjugate KW - Gretchen Hagen 3 KW - Indole-3-acetic acid KW - Indole-3-acetyl-aspartate KW - Pisum sativum SP - 9 EP - 20 JF - Plant physiology and biochemistry : PPB JO - Plant Physiol Biochem VL - 107 N2 - Phytohormone conjugation is one of the mechanisms that maintains a proper hormonal homeostasis and that is necessary for the realization of physiological responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases convert indole-3-acetic acid (IAA) to IAA-amino acid conjugates by ATP-dependent reactions. IAA-aspartate (IAA-Asp) exists as a predominant amide conjugate of auxin in pea tissues and acts as an intermediate during IAA catabolism. Here we report a novel recombinant indole-3-acetic acid-amido synthetase in Pisum sativum. In silico analysis shows that amino acid sequence of PsGH3 has the highest homology to Medicago truncatula GH3.3. The recombinant His-tag-PsGH3 fusion protein has been obtained in E. coli cells and is a soluble monomeric polypeptide with molecular mass of 69.18 kDa. The PsGH3 was purified using Ni(2+)-affinity chromatography and native PAGE. Kinetic analysis indicates that the enzyme strongly prefers IAA and L-aspartate as substrates for conjugation revealing Km(ATP) = 0.49 mM, Km(L-Asp) = 2.2 mM, and Km(IAA) = 0.28 mM. Diadenosine pentaphosphate (Ap5A) competes with ATP for catalytic site and diminishes the PsGH3 affinity toward ATP approximately 1.11-fold indicating Ki = 8.5 μM. L-Tryptophan acts as an inhibitor of IAA-amido synthesizing activity by competition with L-aspartate. Inorganic pyrophosphatase (PPase) hydrolyzing pyrophosphate to two phosphate ions, potentiates IAA-Asp synthetase activity of PsGH3. Our results demonstrate that PsGH3 is a novel enzyme that is involved in auxin metabolism in pea seeds. SN - 1873-2690 UR - https://www.unboundmedicine.com/medline/citation/27235647/Cloning_and_biochemical_characterization_of_indole_3_acetic_acid_amino_acid_synthetase_PsGH3_from_pea_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0981-9428(16)30201-7 DB - PRIME DP - Unbound Medicine ER -