Enzyme activity of LOG as a cytokinin nucleoside 5′-monophosphate phosphoribohydrolase was measured by a method described in an earlier study (Kurakawa et al., 2007) with minor modifications. For conversion analysis, recombinant protein activity was measured by incubating 6 μg of recombinant LOG in a reaction mixture (50 mM Tris, 1 mM MgCl2, 1 mM dithiothreitol, pH 7) with 20 μM substrate (iPRMP or tZRMP), at 30°C for 2 h. The reaction was stopped with three volumes of acetone and stored at −80°C for 30 min. Insoluble material was removed by centrifugation at 15,000 × g for 15 min, at 4°C, and the supernatant was dried using a speed vac (Labconco Corp., MO, United States) at 45°C. The resulting material was dissolved in 1% acetic acid. Cytokinins were separated using UHPLC (Waters) on a Acquity BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters) at a flow rate of 0.3 ml min–1 with gradient of solvent A (1% acetic acid) and solvent B (acetonitrile) according to the following gradient profile: 0 min, 99% A + 1% B; 1 min, 99% A + 1% B; 1.2 min, 93% A + 7% B; 4 min, 90% A + 10% B; 11 min, 60% A + 40% B; 13.50 min, 50% A + 50% B, 15 min 99% A + 1% B. The column temperature was maintained at 40°C, and samples were maintained at 8°C. This experiment had two replicates each and was repeated three times to check repeatability. Cytokinins were separated and monitored by their absorbance at a wavelength of 270 nm. Samples and standards were dissolved in 1% acetic acid. For the determination of kinetic parameters of CvarLOG1 for iPRMP and tZRMP substrates, 4 μg of recombinant LOG was used with various concentrations of substrates. GraphPad Prism was used to calculate the kinetics.

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