Publications

• A mycobacterial cyclic AMP phosphodiesterase that moonlights as a modifier of cell wall permeability.

  Podobnik M., Tyagi R., Matange N., Dermol U., Gupta A.K., Mattoo R., Seshadri K., Visweswariah S.S.

  Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP ... >> More

  Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning. << Less

  J. Biol. Chem. 284:32846-32857(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of the cpdA gene encoding cyclic 3',5'-adenosine monophosphate phosphodiesterase in Escherichia coli.

  Imamura R., Yamanaka K., Ogura T., Hiraga S., Fujita N., Ishihama A., Niki H.

  We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC). The expression of beta-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbor ... >> More

  We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC). The expression of beta-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene. Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells. Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene. We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP. In addition, we showed that the CpdA protein hydrolyzed cAMP to 5'-adenosine monophosphate and that its activity was activated by iron. Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase. << Less

  J. Biol. Chem. 271:25423-25429(1996) [PubMed] [EuropePMC]

• Comparison of enzymatic characterization and gene organization of cyclic nucleotide phosphodiesterase 8 family in humans.

  Gamanuma M., Yuasa K., Sasaki T., Sakurai N., Kotera J., Omori K.

  Full-length cDNAs of human cyclic nucleotide phosphodiesterase 8B (PDE8B) were isolated. Enzymatic characteristics of a dominant variant encoding a protein of 885 residues (PDE8B1) were compared with those of PDE8A1. The recombinant PDE8A1 and PDE8B1 proteins of an entire form were produced in bot ... >> More

  Full-length cDNAs of human cyclic nucleotide phosphodiesterase 8B (PDE8B) were isolated. Enzymatic characteristics of a dominant variant encoding a protein of 885 residues (PDE8B1) were compared with those of PDE8A1. The recombinant PDE8A1 and PDE8B1 proteins of an entire form were produced in both cytosolic and membrane fractions of the transfected COS cells. The human PDE8B1 was a high-affinity cAMP-PDE with K(m) value of 101+/-12 nM for cAMP, which is greater than that of PDE8A1 (40+/-1 nM). Relative V(max) value of PDE8A1 was 57+/-8% compared with that of PDE8B1 (100+/-12%). Although PDE8A1 was moderately inhibited by dipyridamole with IC(50) value of 8+/-2 microM, the compound antagonized the PDE8B1 activity at three-fold higher concentration (IC(50)=23+/-2 microM). The human PDE8B gene was composed of 22 exons, spanning over 217 kb. Although overall sequence identity between PDE8A1 and PDE8B1 was 68%, positions of junctions of each exon between the PDE8A1 and PDE8B1 sequences were well matched, indicating evolutionary relatedness of both genes. << Less

  Cell. Signal. 15:565-574(2003) [PubMed] [EuropePMC]

• The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis.

  Shenoy A.R., Sreenath N., Podobnik M., Kovacevic M., Visweswariah S.S.

  Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclase ... >> More

  Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism. << Less

  Biochemistry 44:15695-15704(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of the memory gene dunce of Drosophila melanogaster.

  Qiu Y.H., Chen C.-N., Malone T., Richter L., Beckendorf S.K., Davis R.L.

  The dunce (dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility. The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4.2 to 9.6 kb ... >> More

  The dunce (dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility. The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases or base-pairs). To characterize these RNAs and to understand their sequence heterogeneity, we isolated and analyzed 29 new and independent cDNA clones representing the dnc RNAs. Restriction mapping, hybridization analysis and sequence determination of these cDNA clones and the corresponding genomic exons resolved these into six different classes. Exons defined by the cDNA clones are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites: two of these were mapped by parallel S1-nuclease and primer extension experiments. In addition, some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence. Altogether, the results indicate that the size and sequence heterogeneity of dnc transcripts results from transcription initiation at multiple sites, alternative splicing, and processes which generate different 3' ends. The existence of multiple protein products is suggested by the alternative use of exons which code for portions of the open reading frame. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons and internal differences arising from the optional inclusion of a 39 base-pair exon and from the alternative use of two 3' splice sites separated by six base-pairs. Expression of a cDNA clone in yeast containing a large portion of the open reading frame produced cAMP PDEase activity identical in properties to the Drosophila enzyme affected by the dnc mutation. The results suggest that the remarkable structural complexity of dnc may reflect an intricate control of the spatial and/or temporal expression of various isoforms of cAMP PDEase. << Less

  J. Mol. Biol. 222:553-565(1991) [PubMed] [EuropePMC]

• cUMP hydrolysis by PDE3A.

  Berrisch S., Ostermeyer J., Kaever V., Kaelble S., Hilfiker-Kleiner D., Seifert R., Schneider E.H.

  As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUM ... >> More

  As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a K _M value of ~143 μM and a V _max value of ~42 μmol/min/mg. PDE3A hydrolyzed cAMP with a K _M value of ~0.7 μM and a V _max of ~1.2 μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (K _i = 57 nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions. << Less

  Naunyn Schmiedebergs Arch. Pharmacol. 390:269-280(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• In vitro and in vivo characterization of the Pseudomonas aeruginosa cyclic AMP (cAMP) phosphodiesterase CpdA, required for cAMP homeostasis and virulence factor regulation.

  Fuchs E.L., Brutinel E.D., Klem E.R., Fehr A.R., Yahr T.L., Wolfgang M.C.

  Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthe ... >> More

  Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthesis and degradation. In the opportunistic human pathogen Pseudomonas aeruginosa, cAMP is a key regulator of virulence gene expression. To better understand the role of cAMP homeostasis in this organism, we identified and characterized the enzyme CpdA, a putative cAMP phosphodiesterase. We demonstrate that CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. Deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. Further, we demonstrate that the cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression. << Less

  J. Bacteriol. 192:2779-2790(2010) [PubMed] [EuropePMC]