Rubisco

The same, commercially obtained spinach Rubisco (Sigma-Aldrich; catalogue # R8000; S1 Fig; specific activity 0.05 units / mg) was inactivated by dissolving in 10 mM HEPES buffer pH 7.9 containing 100 μM EDTA and dialyzing twice against 2 L of ITC buffer (10 mM HEPES buffer pH 7.9) using a 3,500 MWCO dialysis membrane. A low volume Nano ITC instrument (TA Instruments) with a cell volume of 190 μL was used for this study. Stock solutions of RuBP (100 mM) and (+)-ABA (10 mM) were diluted to the working concentration using ITC buffer. The ITC experiments were performed at 20°C. Rubisco, ligands and buffer were equilibrated at 20°C and degassed before the experiment. A 50 μL syringe was used to deliver 19 injections (2.5 μL each) of the ligands (0.75 mM RuBP or 5 mM (+)-ABA) into the cell containing either ITC buffer or Rubisco (39.42 μM). The first injection of 0.71 μL was excluded from data processing. A stir rate of 200 rpm was used to mix the reactants continuously. The data were processed using the NanoAnalyze software (TA Instruments) using the ‘Independent binding’ model.

The following reagents were obtained from Sigma-Aldrich: glucose, 1,3-propanediol, phenylboronic acid, cycloheximide, 23BD, acetoin, pyruvate, PEP, 6PG, G6P, F6P, Ru5P, R15P, ATP, dithiothreitol (DTT), NADH, NADPH, NADP⁺, phosphocreatine, RuBisCO, pyruvate kinase, lactate dehydrogenase, G6P dehydrogenase, 3PGA kinase, GAPDH and creatine kinase. U-¹³C glucose and ¹³C-NaHCO₃ were obtained from Cambridge Isotope Laboratories. IPTG and chloramphenicol were obtained from Fischer Scientific. Gentamycin was purchased from Teknova. Spectinomycin was purchased from MP Biomedicals. Kanamycin was purchased from IBI Scientific. Phusion polymerase was purchased from New England Biolabs. All oligonucleotide synthesis and DNA sequencing were performed by Eurofins MWG Operon Inc.