RRC ID 6662
Author Kuwayama H, Snippe H, Derks M, Roelofs J, Van Haastert PJ.
Title Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium.
Journal Biochem J
Abstract 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.
Volume 353(Pt 3)
Pages 635-44
Published 2001-2-1
DOI 10.1042/0264-6021:3530635
PMID 11171061
PMC PMC1221610
MeSH 1-Methyl-3-isobutylxanthine / pharmacology 3',5'-Cyclic-GMP Phosphodiesterases* / chemistry 3',5'-Cyclic-GMP Phosphodiesterases* / genetics 3',5'-Cyclic-GMP Phosphodiesterases* / metabolism* Amino Acid Sequence Animals Base Sequence Catalytic Domain Cations, Divalent Cloning, Molecular Cyclic GMP / analogs & derivatives* Cyclic GMP / metabolism* DNA, Recombinant Dictyostelium / enzymology* Escherichia coli / genetics Gene Expression Regulation, Enzymologic / drug effects Molecular Sequence Data RNA, Messenger / genetics Sequence Homology, Amino Acid
IF 4.097
Times Cited 25
Cellular slime molds G24660