Identification of 2H phosphoesterase superfamily proteins with 2′-CPDase activity

Abstract

The 2H phosphoesterase superfamily (2H family) proteins are widely conserved among organisms. The 2H family is classified into several subgroups, including YjcG-like proteins whose enzymatic activity has not been reported. In the present study, we found that two YjcG-like proteins (Staphylococcus aureus SA0873 and Bacillus subtilis YjcG) have 2′-CPDase activity that hydrolyzes a 2′,3′-cyclic nucleotide, thereby producing a nucleotide with a 3′-phosphate. The SA0873 protein selectively hydrolyzes a 2′,3′-cyclic nucleotide with a purine base. Four SA0873 mutant proteins (H34A, T36A, H115A, and T117A), in which alanine was substituted for amino acid residues in the HxT/Sx motifs that are conserved in the 2H family, abolished the 2′-CPDase activity. Comparison of three-dimensional structures between the YjcG-like proteins with 2′-CPDase activity and another 2H family subgroup, LigT/2′-5′ RNA ligase-like proteins with 3′-CPDase activity, revealed that the orientation of the substrate binding pocket is reversed between the two groups. Our findings revealed that YjcG-like proteins not only have a substrate-binding pocket different from that of LigT/2′-5′ RNA ligase-like proteins, but they also have 2′-CPDase activity.