Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum.
A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.
Meima
Marcel E
ME
School of Life Sciences, University of Dundee, MSI/WTB complex, Dow Street, United Kingdom.
Weening
Karin E
KE
Schaap
Pauline
P
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004023:Dictyostelium / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D005091:Exons; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D015032:Zinc / Q000378:metabolism
D018076:DNA, Complementary; D008670:Metals; D011993:Recombinant Fusion Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D008345:Manganese; D012313:RNA; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium; D015032:Zinc