Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea
Copyright © 2016 Elsevier Masson SAS. All rights reserved.
| Veröffentlicht in: | Plant physiology and biochemistry : PPB. - 1991. - 107(2016) vom: 27. Okt., Seite 9-20 |
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| Weitere Verfasser: | , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2016
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| Zugriff auf das übergeordnete Werk: | Plant physiology and biochemistry : PPB |
| Schlagworte: | Journal Article Auxin conjugate Gretchen Hagen 3 Indole-3-acetic acid Indole-3-acetyl-aspartate Pisum sativum Dinucleoside Phosphates Enzyme Inhibitors Indoleacetic Acids Plant Proteins mehr... |
| Zusammenfassung: | Copyright © 2016 Elsevier Masson SAS. All rights reserved. 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 |
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| Beschreibung: | Date Completed 27.03.2017 Date Revised 09.01.2024 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1873-2690 |
| DOI: | 10.1016/j.plaphy.2016.05.031 |