Activity assays performed with thin layer chromatography (TLC) TLC conditions were similar to the procedure described by Marini and Ipata (Marini & Ipata, 2007). Polyethyleneimine impregnated cellulose TLC plates (Flexible TLC Plates, Cellulose PEI F-254 from Selecto Scientific, 20 x 20 cm) were rinsed with 10 % NaCl (w/v), then rinsed twice with deionized water (Milli-Q, Millipore), and finally air dried. Next, the plates were cut into four 10 x10 cm squares. A typical reaction mixture contained 2.3 mM of a nucleoside triphosphate, 20 mM MgCl 2 , 50 mM CAPS, pH 10.2, and 0.01 mg/mL of TM0159. Each reaction mixture was incubated in a water bath at 40°C, 50°C, and 60°C. The reaction samples (2 µL) were spotted at the following time intervals: 5, 15, 30, 60, and 90 minutes. For reference, the TLC plates were spotted with 2 µL of standard solutions, such as 0.1 mM ITP and 0.1 mM IMP. All spots were eluted with 0.9 M LiCl, dried, and visualized in UV light at 254 nm with the help of a UV lamp (UV-LAMP-Spectroline model ENF-240C). To check whether TM0159 is also active at room temperature, an additional reaction was set up (2.3 mM ITP, 20 mM MgCl 2 , 50 mM CAPS, pH 10.2, and 0.01 mg/mL of TM0159) and kept at room temperature for 24 hours. After 24 hours, an aliquot was spotted on the TLC plate and visualized. TM0159 readily catalyzes the conversion of ITP to IMP at elevated temperatures. No conversion was detected at room temperature after 24 hours (Fig. S1). We also did not detect any conversion with the nucleoside triphosphate ATP. Figure S1: Photographs of two TLC plates that were used to monitor ITP hydrolysis catalyzed by TM0159 at different temperatures. Standard solutions (0.1 mM ITP and 0.1 mM IMP) were spotted on the plates as well: Lanes 6 & 13 ITP standard; lanes 7 & 14 IMP standard. Lanes 1-5: reaction temperature 40°C spotted after 5 min (lane 1), 15 min (lane 2), 30 min (lane 3), 60 min (lane 4), and 90 min (lane 5). Lanes 8-12: reaction temperature 50°C spotted after 5, 15, 30, 60, and 90 min. Lanes 15-19: reaction temperature 60°C spotted after 5, 15, 30, 60, and 90 min. Lane 20 contains a reaction sample incubated at room temperature for 24 hours.