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 coliFhlA) 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 ∼500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of ∼1 μM. 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 ∼3 μM. 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.
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Research Article|
September 27 2006
Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi
Rocío Díaz-Benjumea;
Rocío Díaz-Benjumea
1
*Instituto de Biología Experimental, Universidad Central de Venezuela, Box 47069, Caracas, Venezuela
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Sunil Laxman;
Sunil Laxman
1
†Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, U.S.A.
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Thomas R. Hinds;
Thomas R. Hinds
†Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, U.S.A.
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Joseph A. Beavo;
Joseph A. Beavo
†Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, U.S.A.
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Ana Rascón
Ana Rascón
2
*Instituto de Biología Experimental, Universidad Central de Venezuela, Box 47069, Caracas, Venezuela
2To whom correspondence should be addressed (email arascon@strix.ciens.ucv.ve).
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Publisher: Portland Press Ltd
Received:
May 22 2006
Revision Received:
June 13 2006
Accepted:
June 16 2006
Accepted Manuscript online:
June 16 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 399 (2): 305–314.
Article history
Received:
May 22 2006
Revision Received:
June 13 2006
Accepted:
June 16 2006
Accepted Manuscript online:
June 16 2006
Citation
Rocío Díaz-Benjumea, Sunil Laxman, Thomas R. Hinds, Joseph A. Beavo, Ana Rascón; Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi. Biochem J 15 October 2006; 399 (2): 305–314. doi: https://doi.org/10.1042/BJ20060757
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