A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi, which interacts with the structural flagellar protein paraflagellar rod.
Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires 1428, Argentina.
Maximiliano A
MA
D'Angelo
Andrea E
AE
Montagna
Santiago
S
Sanguineti
Héctor N
HN
Torres
Mirtha M
MM
Flawiá
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D019281:Dimerization; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014349:Trypanosoma cruzi / Q000201:enzymology*
D017931:DNA Primers; D015800:Protozoan Proteins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium