Variations in the enzymatic activity of S1-type nucleases results from differences in their active site structures.

S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn2+-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities. S1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay. We discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues. Our findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases. Our findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family. • The class of non‑zinc-dependent S1-like nucleases is widely distributed among different phylogenetic groups. • The active sites of S1-like nucleases are highly vulnerable to changes in activity. • The individual requirements of S1-like nucleases are determined by the composition of non-conserved active site residues. • S1-like nuclease family members can adapt to varied physiological conditions and thus contribute to a variety of functions. • The biotechnological applications of S1-like nucleases must take into account the wide range of their activities. [ABSTRACT FROM AUTHOR]