With the initial aim in mind to produce 5'-NTP analogs, we evaluated whether TmNMPKs accept nucleotide analogs as substrates (Figure , Figure -S7). Therefore, enzyme reactions with ATP as phosphate donor were run for 19 h at cytarabine phosphate (araCMP) were tested as substrates for the TmNMPKs. As NMPs were not available for pyrimidine analogs and for arabinofuranosylguanine (araGuo), we screened the TmNMPKs in cascade reactions with the wide spectrum NKs Drosophila melanogaster deoxynucleoside kinase (DmdNK) or human uridine-cytidine kinase 2 (HsUCK2, NK15) for the phosphorylation of 5-bromouridine (5-Br-Urd), 5-F-dUrd, arabinofuranosyluracil (araUrd) and araGuo. To enhance the synthesis of the NTPs , an ATP regeneration system based on TmAcK was integrated. All modified substrates tested were at least accepted by one TmNMPK (Figure , Figure -S7), which indicates their high potential for the in vitro synthesis of nucleotide analogs. So far, mainly NMPKs from human and pathogenic organisms were studied for their ability to phosphorylate nucleotide analogs, since they are responsible for the therapeutic efficiency of nucleoside/nucleotide drugs . Apart from that, there are only few reports about nucleotide analog synthesis involving NMPKs, including the synthesis of 3'-F-(d)ATP and 3'F-(d)GTP by yeast NMPKs , several 2'-/3'-modified NMPs by calf thymus NMPKs , 5-F-CTP, 5-F-UTP by Escherichia coli NMPKs , as well as adenosine 5'-(O-1-thiotriphosphate), adenosine 5'-(O-2thiotriphosphate) and ribavirin triphosphate by rabbit muscle AMPK . Notably, to our best knowledge we demonstrated here for the first time the acceptance of nucleotide analogs by thermostable NMPKs. -1 NMPK and 0.02 mg mL -1 AcK. Cascade reactions were incubated for 19 h at 37 °C and analyzed by TLC or HPLC (see Figure and). (+) < 10% NDP formation, (++) NDP formation between 10% and 50%, + < 10% NTP formation, ++ NTP formation between 10% and 50%, +++ NTP formation > 50% For TmAMPK NTP formation was observed for all AMP analogs (Figure ). Interestingly, although CMP and UMP are natural substrates, araUMP was accepted but not araCMP. Furthermore, neither the other UMP analogs nor araGMP were phosphorylated. The preference of AMP analogs is in good agreement with the data for other AMPKs . In contrast to the phosphorylation of deoxycytidine analogs like araCMP , the acceptance of araUMP was not described before for AMPKs.