Systematic characterization of the ADP-ribose pyrophosphatase family in the Cyanobacterium Synechocystis sp. strain PCC 6803.
We have characterized four putative ADP-ribose pyrophosphatases Sll1054, Slr0920, Slr1134, and Slr1690 in the cyanobacterium Synechocystis sp. strain PCC 6803. Each of the recombinant proteins was overexpressed in Escherichia coli and purified. Sll1054 and Slr0920 hydrolyzed ADP-ribose specifically, while Slr1134 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 Sll1054 is a bacterial type, while the other three and Slr0787, 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.
Graduate School of Science and Engineering, Ehime University, Bunkyo-cho, Matsuyama, Japan.
Kenji
K
Okuda
Hidenori
H
Hayashi
Yoshitaka
Y
Nishiyama
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D046939:Synechocystis / Q000145:classification / Q000201:enzymology*
D007527:Isoenzymes; D011755:Pyrophosphatases; C019620:ADPribose pyrophosphatase