| Autor: |
Galka MM; National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada., Rajagopalan N; National Research Council of Canada, Saskatoon, Saskatchewan, Canada., Buhrow LM; National Research Council of Canada, Saskatoon, Saskatchewan, Canada., Nelson KM; National Research Council of Canada, Saskatoon, Saskatchewan, Canada., Switala J; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada., Cutler AJ; National Research Council of Canada, Saskatoon, Saskatchewan, Canada., Palmer DR; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada., Loewen PC; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada., Abrams SR; National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada., Loewen MC; National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. |
| Abstrakt: |
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. |
