Decapping scavenger enzymes (DcpSs) are important players in mRNA degradation machinery and conserved in eukaryotes. Importantly, human DcpS is the recognized target for spinal muscular atrophy (SMA) and acute myeloid leukemia (AML) therapy, and has recently been connected to development of intellectual disability. Most recombinant DcpSs used in biochemical and biophysical studies are prepared as tagged proteins, with polyhistidine (His-tag) at the N-terminus or C-terminus. Our work is the first report on the parallel characterization of three versions of DcpSs (native and N- or C-terminally tagged) of three species (humans, and ). The native forms of all three enzymes were prepared by N-(His) tag cleavage. Protein thermal stability, measured by differential scanning fluorimetry (DSF), was unaffected in the case of native and tagged versions of human and DcpS; however, the melting temperature () of DcpS of was significantly influenced by the presence of the additional N- or C-tag. To investigate the impact of the tag positioning on the catalytic properties of DcpS, we tested the hydrolytic activity of native DcpS and their His-tagged counterparts toward cap dinucleotides (mGpppG and mGpppG) and mGDP. The kinetic data indicate that dinucleotide substrates are hydrolyzed with comparable efficiency by native human and DcpS and their His-tagged forms. In contrast, both His-tagged DcpSs exhibited higher activity toward mGpppG than the native enzyme. mGDP is resistant to enzymatic cleavage by all three forms of human and nematode DcpS.Copyright © 2020 American Chemical Society.