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Appendix S3: Enzymatic assays
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All the enzymatic assays were run in triplicates with previously purified
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recombinant enzyme, in 96 wells plates (Greiner BioOne, Monroe, NC) in a 100 µL
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reaction mix containing, 0.1 M PBS pH 6.8, 0.025g/L BSA, 1 mM AcCoA, 0.5 µg of
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enzyme and variable concentrations of substrate depending on the experiment. The
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mix without enzyme was first held for 10 min at the desired temperature before
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enzyme was added. After 10 min incubation, 150 µL of a stop solution (6 M guanidine
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hydrochloride, 12 mM EDTA, 4 mM DTNB 5,5'-dithiobis-(2-nitrobenzoic acid), 0.1 M
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Tris pH 6.8) was added to the reaction mix and incubated 5 min before lecture on a
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spectrophotometer plate reader. The choice of buffer pH and molarity, and AcCoA
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and enzyme concentrations, was dictated by previous validation studies, making sure
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that initial velocity could be calculated. Saturation curves were realized with 8
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different concentrations of substrate at the desired temperatures as indicated in the
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results and figure legends. Saturation curves of the temperature assay are provided
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for the 5 species investigated in Figure S1. The impact of enzyme denaturation was
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investigated by placing the enzyme solutions at 45°C or at 65°C for 5, 10, 20, 40, and
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60 min, after what activity was measured as described above, using 20 mM
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tryptamine and a 20°C incubation temperature.
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