2a and c), while for the GST fusion proteins, FpdNK + GST and PddNK + GST, it was around 50 kDa, because of the GST part (Fig. 2b and d). Kinetic parameters were determined for purified recombinant PdTK1, PddNK + GST, FpTK1, and FpdNK + GST (Table 1).

The activity was measured at 37 °C, except for FpTK1 and PdTK1, which were tested at 21 °C. All enzyme reactions followed classical Michaelis–Menten kinetics. The FpTK1 specifically phosphorylated dT and dU, having dT as the preferred substrate, because the Km for dT was 2.2 μM, which is ~ 64 times lower signaling pathway than for dU. The apparent maximal velocity (Vm-app) was 5.78 U mg−1 for dT and 4.64 U mg−1 for dU (Table 1, Fig. S2a and b). Similarly, also PdTK1 preferred dT as substrate over dU, with Km for dT being 32 μM, which is ~ 27 times lower than for dU. The Vm-app for dT was 3.43 and 2.11 U mg−1 for dU (Table 1, Fig. S2e and f). The catalytic efficiency (Vm-app/Km) was manyfold higher for FpTK1 than for PdTK1, and the activity with dT was higher for both enzymes. This indicates that FpTK1 has higher specificity toward dT and dU (Table 1). We could not detect any significant phosphorylation of dA, dC, or dG by either FpTK1 or PdTK1. The FpdNK was able to phosphorylate both dA

and dC, but had dA as the preferred substrate, with the Km for dA being 4.5 times lower than for dC. The catalytic efficiency (Vm-app/Km) was manyfold higher for dA than for dC, indicating that dA was the preferred substrate (Table 1, Fig. S2c and d). Also PddNK preferred dA as substrate over dC, the catalytic efficiency being almost 6-fold higher for dA than for dC (Table 1, Fig. S2g and h). In short, both GSI-IX non-TK1 kinases were much more Rapamycin in vivo specific

for dA than for dC, and none of them was able to phosphorylate dG. Initially, attempts to measure the substrate specificity of pure recombinant PdTK1 and FpTK1 at 37 °C failed; therefore, we decided to determine PdTK1 phosphorylating activity as a function of temperature, by measuring the activity at 500 μM 3H-dT and 2.5 mM ATP, at different temperatures, and with prolonged sampling times. It turned out that the activity of PdTK1 increased with temperature, up to 21 °C, where the highest activity was detected (7.77 ± 1.56 U mg−1); thereafter, the activity decreased with increasing temperature (Fig. 3). Therefore, 21 °C was used for further investigation of PdTK1 and FpTK1. Upon pre-incubating the enzyme at 0 °C for one hour, the obtained activity at 21 °C was considerably lower (0.71 ± 0.04 U mg−1), and after pre-incubation at 37 °C for one hour, the enzyme was irreversibly denatured, because we could not detect any activity at all. One of the approaches to estimate the aquatic bacteria biomass production is the incorporation of 3H-dT into newly synthesized DNA, and it is based on the assumption that all actively growing bacteria can incorporate external dT into DNA (Furhman & Azam, 1982).