title: 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 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.affiliation: Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland. Electronic address: maciejost@umk.pl.
authors.forename: Maciej
authors.initials: M
authors.lastname: Ostrowski
authors.affiliation: Department of Genetics, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Mierek-Adamska
authors.affiliation: Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Porowińska
authors.affiliation: Department of Genetics, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Goc
authors.affiliation: Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Jakubowska
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity / Q000187:drug effects; D014364:Tryptophan / Q000378:metabolism
chemical_list: D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D007210:Indoleacetic Acids; D010940:Plant Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C030737:indoleacetic acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D008025:Ligases
keywords: Auxin conjugate; Gretchen Hagen 3; Indole-3-acetic acid; Indole-3-acetyl-aspartate; Pisum sativum