title: Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.
abstract: Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Marek M
authors.initials: MM
authors.lastname: Galka
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada.
authors.forename: Nandhakishore
authors.initials: N
authors.lastname: Rajagopalan
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada.
authors.forename: Leann M
authors.initials: LM
authors.lastname: Buhrow
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada.
authors.forename: Ken M
authors.initials: KM
authors.lastname: Nelson
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Jacek
authors.initials: J
authors.lastname: Switala
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Cutler
authors.affiliation: Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: David R J
authors.initials: DR
authors.lastname: Palmer
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Loewen
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Abrams
authors.affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Michele C
authors.initials: MC
authors.lastname: Loewen
mesh_terms: D000040:Abscisic Acid / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D001665:Binding Sites; D008969:Molecular Sequence Data; D011485:Protein Binding; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000040:Abscisic Acid; D012273:Ribulose-Bisphosphate Carboxylase