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 phosphodiesterases (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 presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino 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 glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis-Menten kinetics and a higher affinity for cGMP (K(m)=0.22 microM) than for cAMP (K(m)=145 microM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn(2+) is preferred to Mg(2+), whereas Ca(2+) yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of approx. 60 microM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.
Kuwayama
H
H
GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Snippe
H
H
Derks
M
M
Roelofs
J
J
Van Haastert
P J
PJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002413:Cations, Divalent; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism*; D004274:DNA, Recombinant; D004023:Dictyostelium / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D002413:Cations, Divalent; D004274:DNA, Recombinant; D012333:RNA, Messenger; C016276:8-bromocyclic GMP; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine