The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications

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