Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea

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 K m ATP  = 0.49 mM, K m L−Asp  = 2.2 mM, and K m IAA  = 0.28 mM. Diadenosine pentaphosphate (Ap 5 A) competes with ATP for catalytic site and diminishes the PsGH3 affinity toward ATP approximately 1.11-fold indicating K i  = 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.