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

•Recombinant PsGH3 protein catalyzes synthesis of auxin-amino acid conjugates in ATP-dependent manner.•The enzyme strongly prefers indole-3-acetic acid and aspartic acid as substrates for the conjugation.•Diadenosine pentaphosphate competes with ATP during catalytic reaction and inhibits PsGH3 activity.•An exogenous inorganic pyrophosphatase potentiates amidosynthetase activity.

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 Ni2+-affinity chromatography and native PAGE. Kinetic analysis indicates that the enzyme strongly prefers IAA and L-aspartate as substrates for conjugation revealing KmATP = 0.49 mM, KmL−Asp = 2.2 mM, and KmIAA = 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.