Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the ligand for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is encoded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent beta1-beta3 region of the GAF B subdomain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure-function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and biochemically one by one.     

The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain

The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 microm cGMP for half-maximal effective concentration (EC(50)) of a cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we examine whether modifications in the N-terminus of PDE11A4 affect cGMP signalling. The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases (Ser117, Ser168). Phosphorylation of both by cAMP-dependent protein kinase decreased the EC(50) value for cGMP from 72 to 23 microm. Phosphomimetic point mutations (S117D/S167D), which project complete phosphorylation, lowered the EC(50) value to 16 microm. Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC(50) value of 7.6 microm. Truncation by 176 amino acids was required for cGMP EC(50) values to decrease to below 10 microm; a construct truncated by 168 amino acids had an EC(50) value of 224 microm. The decrease in EC(50) values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however. We conclude that N-terminal modifications strongly affect cGMP regulation of hPDE11A4.     

Characterization of the tandem GAF domain of human phosphodiesterase 5 using a cyanobacterial adenylyl cyclase as a reporter enzyme

We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K(D) for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.     

Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.     

Functional chimeras of the phosphodiesterase 5 and 10 tandem GAF domains

The tandem GAF domain of hPDE10A uses cAMP as an allosteric ligand (Gross-Langenhoff, M., Hofbauer, K., Weber, J., Schultz, A., and Schultz, J. E. (2006) J. Biol. Chem. 281, 2841-2846). We used a two-pronged approach to study how discrimination of ligand is achieved in human (h)PDE10A and how domain selection in the phosphodiesterase GAF tandems is determined. First, we examined which functional groups of cAMP are responsible for purine ring discrimination. Changes at the C-6 ring position (removal of the amino group; chloride substitution) and at the N-1 ring position reduced stimulation efficacy by 80%, i.e. marking those positions as decisive for nucleotide discrimination. Second, we generated a GAF tandem chimera that consisted of the cGMP-binding GAF-A unit from hPDE5A1, which signals through cGMP in PDE5, and the GAF-B from hPDE10A1, which signals through cAMP in PDE10. Stimulation of the reporter enzyme exclusively was through the GAF-B domain of hPDE10A1 (EC(50) = 7 mum cAMP) as shown by respective point mutations. The PDE5 GAF-A domain in the chimera did not signal, and its function was reduced to a strictly structural role. Signaling was independent of the origin of the N terminus. Generating 10 additional PDE5/10 tandem GAF chimeras surprisingly demonstrated that the length-conserved linker in GAF tandems between GAF-A and GAF-B played an unforeseen decisive role in intramolecular signaling. Swapping the linker sections between PDE5 and PDE10 GAF tandem domains abrogated signaling completely pointing to specific domain interactions within GAF tandems, which are not visible in the available crystal structures with bound ligands.     

Crystal structure of the GAF-B domain from human phosphodiesterase 10A complexed with its ligand, cAMP

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.     

GAF domains: two-billion-year-old molecular switches that bind cyclic nucleotides

GAF domains represent one of the largest families of small-molecule binding units present in nature. The first mammalian GAF domains discovered were the cGMP-binding regulatory domains of several cyclic nucleotide phosphodiesterases (PDEs). The crystal structure of the PDE2A GAF domains has provided our first look at the architecture of the binding site for the second messenger cGMP. The topology of this site differs greatly from all other previously determined cyclic nucleotide binding sites. In PDE2A, cGMP binds to a well-defined pocket in one of the two GAF domains that is analogous to the ligand-binding pocket of the distantly related PAS domains of photoactive yellow protein and FixL. The consensus cGMP-binding motif suggests strongly that only certain GAF domains will bind cGMP. Although the detailed mechanism for how cGMP binding to the GAF domain regulates catalysis remains to be determined, recent data from a GAF domain-containing cAMP-stimulated adenylyl cyclase from Anabaena suggest a mechanism conserved across two billion years of evolution. Because of their unique ligand-binding topologies, the GAF domains of PDEs are likely to offer good new targets for rational drug design.     

Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory gamma subunit (Pgamma), known to directly inhibit PDE6 catalysis, was increased approximately 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pgamma to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pgamma1-60, Pgamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pgamma1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pgamma-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.     

Trypanosome cyclic nucleotide phosphodiesterase 2B binds cAMP through its GAF-A domain

Trypanosoma brucei, the causative agent of sleeping sickness in humans and livestock, expresses at least three cAMP-specific class I phosphodiesterases (PDEs), all of which are essential for survival of the parasite. These PDEs have either one or two N-terminal GAF domains, which in other proteins function as signaling domains. However, neither the functional roles nor ligands for these domains in trypanosome PDEs are known. The present study shows that TbPDE2B, which contains two tandem GAF domains, binds cAMP with high affinity through its GAF-A domain. A purified recombinant N terminus + GAF-A domain binds cAMP with an affinity (Ki) of approximately 16 nM. It also binds cGMP but with a 15-fold lower affinity of approximately 275 nM. The TbPDE2B holoenzyme has a somewhat lower affinity (approximately 55 nM) for cAMP but a greatly lower affinity (approximately 10 microM) for cGMP. This suggests that both the selectivity and affinity for a ligand can be determined not only by the nature of the binding domain but also by the adjacent domains. Additionally, binding of cAMP to the holoenzyme showed positive cooperativity, with a Hill coefficient value of 1.75. However, binding of cGMP to the holoenzyme did not show any cooperativity, suggesting differences in the conformational changes caused by binding of these two cyclic nucleotides with the protein. Point mutation of a key predicted binding site residue (T317A) resulted in a complete loss of high affinity cAMP binding. This mutation increased the apparent Km of the mutant enzyme for substrate without altering the Vmax. A truncated catalytic domain construct of TbPDE2B also exhibited an increased Km, strongly suggesting that cAMP binding to the GAF-A domain can regulate TbPDE2B by allowing the full activity of the enzyme to be expressed. These properties of the GAF-A domain of TbPDE2B thus suggest that it could be a new target for anti-trypanosomal drugs.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.     

Activation of PDE10 and PDE11 phosphodiesterases

The most recently identified cyclic nucleotide phosphodiesterases, PDE10 and PDE11, contain a tandem of so-called GAF domains in their N-terminal regulatory regions. In PDE2 and PDE5, the GAF domains mediate cGMP stimulation; however, their function in PDE10 and PDE11 remains controversial. Although the GAF domains of PDE10 mediate cAMP-induced stimulation of chimeric adenylyl cyclases, cAMP binding did not stimulate the PDE10 holoenzyme. Comparable data about cGMP and the PDE11 GAF domains exist. Here, we identified synthetic ligands for the GAF domains of PDE10 and PDE11 to reduce interference of the GAF ligand with the catalytic reaction of PDE. With these ligands, GAF-mediated stimulation of the PDE10 and PDE11 holoenzymes is demonstrated for the first time. Furthermore, PDE10 is shown to be activated by cAMP, which paradoxically results in potent competitive inhibition of cGMP turnover by cAMP. PDE11, albeit susceptible to GAF-dependent stimulation, is not activated by the native cyclic nucleotides cAMP and cGMP. In summary, PDE11 can be stimulated by GAF domain ligands, but its native ligand remains to be identified, and PDE10 is the only PDE activated by cAMP.     

PDE5 is converted to an activated state upon cGMP binding to the GAF A domain

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.     

The regulatory tandem domains of human phosphodiesterases 1 and 4 regulate a cyanobacterial adenylyl cyclase

Human phosphodiesterase 1 is regulated by a tandem of N-terminal calmodulin/Ca(2+)-binding domains. We grafted the tandems from hPDE1A3 and -B1 onto the cyanobacterial adenylyl cyclase CyaB1 thus replacing an intrinsic tandem GAF-domain. Cyclase activity was stimulated by Ca(2+)/calmodulin 1.9 to 4.4-fold, i.e. similarly as reported for hPDE1 regulation. hPDE4 long isoforms are activated by phosphorylation of a serine located in a conserved RRESF motif in a tandem of N-terminal upstream-conserved regions (UCR). We grafted the UCR tandems from hPDE4A4, -B1, and -D3 onto the CyaB1 cyclase as a reporter enzyme. Activity was enhanced 1.4 to 4.5-fold by respective phosphomimetic (S/D) point mutations. Similarly, cyclase activity was increased 2.5-fold by phosphorylation of the chimera with the PDE4D3 UCR tandem by cAMP-dependent protein kinase. We propose a common mechanism of activation in mammalian phosphodiesterases containing N-terminal tandem regulatory domains. A downstream region is suggested to alternate between random and ordered conformations and to enable switching between inactive, the catalytic domain occluding PDE homodimers and active monomeric PDE catalytic domains.     

A FYVE-containing unusual cyclic nucleotide phosphodiesterase from Trypanosoma cruzi

Cyclic-nucleotide-specific phosphodiesterases (PDEs) are key players in the intracellular signaling pathways of the important human pathogen Trypanosoma cruzi. We report herein the identification of an unusual PDE from this protozoal organism. This enzyme, TcrPDEC, is a member of the class I PDEs, as determined from the presence of a characteristic signature sequence and from the conservation of a number of functionally important amino acid residues within its catalytic domain. Class I PDEs include a large number of PDEs from eukaryotes, among them all 11 human PDE families. Unusually for an enzyme of this class, TcrPDEC contains a FYVE-type domain in its N-terminal region, followed by two closely spaced coiled-coil domains. Its catalytic domain is located in the middle of the polypeptide chain, whereas all other class I enzymes contain their catalytic domains in their C-terminal parts. TcrPDEC can complement a PDE-deficient yeast strain. Unexpectedly for a kinetoplastid PDE, TcrPDEC is a dual-specificity PDE that accepts both cAMP and cGMP as its substrates.     

Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE). Identification of GAF domains in PDE alphabeta subunits and distinct domains in the PDE gamma subunit involved in stimulation of cGMP binding to GAF domains

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Palphabeta) and two identical regulatory subunits (Pgamma). Biochemical studies have shown that amphibian Palphabeta has high affinity, cGMP-specific, non-catalytic binding sites and that Pgamma stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pgamma-depleted membrane-bound PDE (20-40% Pgamma still present), a single type of cGMP-binding site with a relatively low affinity (K(d) approximately 100 nm) was observed, and addition of Pgamma increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pgamma reduced its ability to stimulate cGMP binding. Among these, the site involved in Pgamma phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pgamma inhibition of the cGMP hydrolytic activity of Palphabeta. In addition, the Pgamma concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Palphabeta heterodimer contains two spatially and functionally distinct types of Pgamma-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Palphabeta-Pgamma interaction in which Pgamma, by binding to one of the two sites in Palphabeta, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.     

The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change

The photoreceptor phosphodiesterase (PDE6) regulates the intracellular levels of the second messenger cGMP in the outer segments of cone and rod photoreceptor cells. PDE6 contains two regulatory GAF domains, of which one (GAF A) binds cGMP and regulates the activity of the PDE6 holoenzyme. To increase our understanding of this allosteric regulation mechanism, we present the 2.6A crystal structure of the cGMP-bound GAF A domain of chicken cone PDE6. Nucleotide specificity appears to be provided in part by the orientation of Asn-116, which makes two hydrogen bonds to the guanine ring of cGMP but is not strictly conserved among PDE6 isoforms. The isolated PDE6C GAF A domain is monomeric and does not contain sufficient structural determinants to form a homodimer as found in full-length PDE6C. A highly conserved surface patch on GAF A indicates a potential binding site for the inhibitory subunit Pgamma. NMR studies reveal that the apo-PDE6C GAF A domain is structured but adopts a significantly altered structural state indicating a large conformational change with rearrangement of secondary structure elements upon cGMP binding. The presented crystal structure will help to define the cGMP-dependent regulation mechanism of the PDE6 holoenzyme and its inhibition through Pgamma binding.     

Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.