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. 2007 Feb 15;402(1):153-61.
doi: 10.1042/BJ20061153.

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

Sonya Bader  1 , Arjan Kortholt, Peter J M Van Haastert
Affiliations

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

Sonya Bader et al. Biochem J. .

Abstract

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.

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Figures

Figure 1
Figure 1. The PDE family of D. discoideum
(A) Seven PDEs have been recognized and identified in the completed genome of D. discoideum. The enzymes belong to the class I enzymes prevalent in higher eukaryotes, or to the class II enzymes that are present in lower eukaryotes and some bacteria. Based on bootstrap data of phylogenetic cluster analysis of type II enzymes, the class II enzymes are recognized as catalytic PDEs or catalytic metallo-β-lactamases. The localization and activity of the seven enzymes are regulated by signal sequences, transmembrane segments, a response regulator domain or cNB domains. (B) Cluster analysis of the catalytic domains of class II enzymes from prokaryotes and lower eukaryotes. All nodes have bootstrap values above 70%, except for the nodes indicated by the small white ovals. Bootstrap values of the inner nodes are indicated. These bootstrap values and the length of the catalytic domain suggest two main groups, metallo-β-lactamases and PDEs; the latter may consist of two subgroups. The species abbreviations, gene identifiers and taxonomy are: Dh, Debaryomyces hansenii, gi|50424793, Eukaryota, Fungi; Cal, Candida albicans, gi|7548341, Eukaryota, Fungi; Kl, Kluyveromyces lactis, gi|50307193, Eukaryota, Fungi; Eg, Eremothecium gossypii, gi|44984787, Eukaryota, Fungi; Cg, Candida glabrata, gi|49526440, Eukaryota, Fungi; Sc, Saccharomyces cerevisiae, gi|6321189, Eukaryota, Fungi; Yl, Yarrowia lipolytica, gi|50554561, Eukaryota, Fungi; Nc, Neurospora crassa, gi|85086599, Eukaryota, Fungi; Sp, Schizosaccharomyces pombe, gi|3581909, Eukaryota, Fungi; Dd, D. discoideum; DdPDE1, gi|84080, DdPDE5, gi|21069535, DdPDE6, gi|66819225, DdPDE7, gi|66805301, Eukaryota, Mycetozoa; Lp, Legionella pneumophila, gi|53754670, Bacteria, Proteobacteria; Pa, Pseudoalteromonas atlantica, gi|76793857, Bacteria, Proteobacteria; Yp, Yersinia pseudotuberculosis, gi|77629947, Bacteria, Proteobacteria; Vf, Vibrio fischeri, gi|59711863, Bacteria, Proteobacteria; Ddβ-lactamase, D. discoideum, gi|66802803, Eukaryota, Mycetozoa; Mm, Magnetospirillum magnetotacticum, gi|23012958, Bacteria, Proteobacteria; Cau, Chloroflexus aurantiacus, gi |76165595, Bacteria, Chloroflexi; Tt, Tetrahymena thermophila; Tt1, gi|89287508, Tt2, gi|89296516, Eukaryota, Alveolata; Li, Leptospira interrogans, gi|45657830, Bacteria, Spirochaetes.
Figure 2
Figure 2. Alignment of DdPDE1 and DdPDE7
The deduced amino acid sequence of DdPDE1 and DdPDE7. Underlined are the predicted signal sequences with cleavage between amino acid 17 and 18 for DdPDE1 and 23 and 24 for DdPDE7. A potential hydrophobic transmembrane segment is indicated in small italic letters. The asterisk indicates a potential GPI anchor.
Figure 3
Figure 3. Initial characterization of DdPDE7
(A) Inhibition by IBMX and DTT of PDE activity at the surface of wild-type AX3 cells and UK7 cells with a deletion of DdPDE1 that had been starved for 5 h. The activity in the absence of inhibitors is set at 100% (2950 fmol/min per 107 cells in AX3 and 61 fmol/min per 107 cells in UK7). The results show that the residual PDE activity exposed on UK7 cells is inhibited strongly by DTT and only partly by IBMX. (B) Over-expression of DdPDE7 in UK7 cells reveal enhanced activity both on the cell surface and secreted in the medium.
Figure 4
Figure 4. Kinetics of DdPDE7
UK7 cells that had been starved for 5 h were incubated with different concentrations of cAMP or cGMP. The hydrolysis was measured after 30 min and expressed as an Eady–Hofstee plot in the main figure and as a Hill plot in the inset. The dotted lines were fitted for Michaelis–Menten kinetics using linear regression, while the straight lines were fitted for negative co-operativity using the Hill equation. The deduced kinetic constants for the Hill equation are, for cAMP apparent Km=12.5 μm, Vmax=28 pmol/min per 107cells, Hill coefficient=0.79, and for cGMP, apparent Km=36 μm, Vmax=72 pmol/min per 107cells and Hill coefficient=0.9.
Figure 5
Figure 5. DdPDE7 activity during development
(A), UK7/regA− cells were starved on plates for the times indicated, harvested, washed and assayed for cell surface cAMP hydrolysis using 10 nM [3H]cAMP. Cell aggregation was observed at 6 h of development, and tight aggregates and slugs were observed at 9 h; development was arrested at this stage. (B) Activity of DdPDE1, DdPDE4 and DdPDE7 during development, deduced from (A) for DdPDE7, and from Figure 6 in Bader et al. [28] for DdPDE1 and DdPDE4.
Figure 6
Figure 6. Contribution of the seven PDEs to the hydrolysis of three cyclic nucleotide pools
(A) Extracellular cAMP during aggregation; (B) extracellular cAMP in slugs; (C) intracellular cAMP during aggregation; (D) intracellular cGMP during aggregation. The activity of each enzyme was calculated at different substrate concentrations using the equations presented in the Experimental procedures section and the kinetic constants as presented in Table 1. From these activities we calculated the half-life of degradation of the indicated substrate concentration, and the relative contribution of the participating enzymes in the degradation of substrate. The arrows indicate the range of substrate concentrations that have been measured in vivo during cell stimulation.
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