Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodie sterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the pres ence of at least one cGMP-specific phosphodiesterase (PDE) that is activate d by cGMP. Using bioinformatics we identified a partial sequence in the Dic tyostelium expressed sequence tag database that shows a high degree of amin o acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60 kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and g lutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis-Menten kinetics and a higher affi nity for cGMP (K-m = 0.22 muM) than for cAMP (K-m = 145 muM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activi ty; Mn2+ is preferred to Mg2+, whereas Ca2+ yields no activity. DdPDE3 is i nhibited by 3-isobutyl-1-methylxanthine with an IC50 of approx. 60 muM. Ove rexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The prope rties of DdPDE3 resemble those of mammalian PDE9, which also shows the high est sequence similarity within the catalytic domains. DdPDE3 is the first c GMP-selective PDE identified in lower eukaryotes.