Systematic characterization of the ADP-ribose pyrophosphatase family in the cyanobacterium Synechocystis sp strain PCC 6803

We have characterized four putative ADP-ribose pyrophosphatases SII1054, SIr0920, SIr1134, and SIr1690 in the cyanobacterium Synechocystis sp. strain PCC 6803. Each of the recombinant proteins was overexpressed in Escherichia coli and purified. SII1054 and Sh-0920 hydrolyzed ADP-ribose specifically, while SIr1134 hydrolyzed not only ADP-ribose but also NADH and flavin adenine dinucleotide. By contrast, Slr1690 showed very low activity for ADP-ribose and had four substitutions of amino acids in the Nudix motif, indicating that Slr1690 is not an active ADP-ribose pyrophosphatase. However, the quadruple mutation of Slr1690, T73G/ I88E/K92E/A94G, which replaced the mutated amino acids with those conserved in the Nudix motif, resulted in a significant (6.1 X 10(2)-fold) increase in the k(cat) value. These results suggest that Slr1690 might have evolved from an active ADP-ribose pyrophosphatase. Functional and clustering analyses suggested that SII1054 is a bacterial type, while the other three and SIr0787, which was characterized previously (Raffaelli et al., FEBS Lett. 444:222-226, 1999), are phylogenetically diverse types that originated from an archaeal Nudix protein via molecular evolutionary mechanisms, such as domain fusion and amino acid substitution.