Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.
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PMID:Calmodulin-like activity in mycobacteria. 166 96

To test the hypothesis that T lymphocytes sensitized to central nervous system (CNS) antigens may quantitatively induce more demyelination in neural tissue than T cells sensitized to non-CNS antigens, we established T cell lines specific for myelin basic protein (MBP) or the purified protein derivative (PPD) of M. tuberculosis. The potential of T cells to cause myelin pathology was determined by measuring the activity of the myelin-associated enzyme 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in organotypic cultures of syngeneic spinal cord after incubation with the T cell lines. The activity of CNPase in neural tissue has been shown to correlate positively with the amount and integrity of CNS myelin. Although both MBP- and PPD-specific T cells caused decreases in CNPase activity, the MBP line caused significantly greater and consistent changes. This finding indicates that T cell-mediated CNS demyelination may be comprised of CNS antigen-specific and CNS non-specific components, the former causing more pathology.
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PMID:Quantitation of antigen-specific T-cell-induced demyelination in vitro. 244 26

It was possible to define the effects of trehalose dimycolate (TDM), a glycolipid extracted from Mycobacterium tuberculosis, on mouse peritoneal macrophages more precisely using endotoxin-free culture conditions. TDM-elicited macrophages, when assayed in vitro in the absence of endotoxin, were unable to limit tumor growth; however, after a short treatment (4 h) with low doses of lipopolysaccharide (LPS; 1-10 ng/ml), they exhibited a strong cytostatic capacity against P815 mastocytoma cells. Thus, TDM injected in vivo did not activate macrophages fully but it primed them to respond in vitro to low doses of LPS, which provided the final stimulus for activation to antitumor competence. Macrophages elicited by an injection of killed group C Streptococci were also in a primed state; in contrast, thioglycollate-elicited macrophages were in a nonreceptive state. Besides LPS, concanavalin A (5 micrograms/ml), MDP (0.2-1 microgram/ml) and the ionophore A23187 (5 microM) can deliver the activation signal to TDM-primed macrophages. Primed macrophages were found to express several biochemical markers previously described as specific for activated macrophages (low levels of alkaline phosphodiesterase and beta-galactosidase, for example) and, although they were not cytotoxic for tumor cells, they had the capacity to release large amounts of H2O2. However, when pulsed by LPS or MDP, primed macrophages responded by further modifications in their metabolism: the rate of glucose consumption and the labeling of glycoproteins by D-[2-3H]mannose were greatly increased and the secretion of a polypeptide of 22 kDa was enhanced. The activation-associated biochemical markers are thus acquired in two steps. The ability to produce activated oxygen species is expressed earlier than the antitumoral activity.
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PMID:Macrophage activation by trehalose dimycolate requirement for an expression signal in vitro for antitumoral activity; biochemical markers distinguishing primed and fully activated macrophages. 300 1

DNA polymerase has been purified approximately 2000-fold from Mycobacterium tuberculosis H37Rv. The purified preparation was homogeneous by electrophoretic criteria and has a molecular weight of 135 000. The purified enzyme resembles Escherichia coli polymerase I in its properties, being insensitive to sulfhydryl drugs and possessing 5',3'-exonuclease activity in addition to polymerase and 3',5'-exonuclease activities. However, it differs from the latter in its sensitivity to higher salt concentration and DNA intercalating agents such as 8-aminoquinoline. The polymerase exhibited maximal activity between 37--42 degrees C and pH 8.8--9.5. The polymerase was stable for several months below 0 degree C. However, the 5',3'-exonuclease activity was more labile. The effects of different metal ions, polyamines and drugs on the polymerase activity are presented.
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PMID:Purification and properties of DNA polymerase from Mycobacterium tuberculosis H37Rv. 678 93

Trehalose diesters (natural 6,6'-trehalose dimycolate from Mycobacterium tuberculosis or synthetic (a 76 carbon atom analogue)), when suspended in water, give stable and well-defined emulsions. These emulsions, injected i.p. in mice significantly limit the growth of P815 syngeneic mastocytoma cells. They elicit macrophages with a cytostatic activity against P815 cells in vitro, strong enough to be expressed at low effector to target ratios (E/T = 1.4) or after a short coincubation period (2 hr). The antitumor potential of these macrophages seems to coincide with their ability to release H2O2 upon pharmacologic triggering. Depressed levels of alkaline phosphodiesterase and beta-galactosidase are proposed as other biochemical markers of cytostatic macrophages.
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PMID:Antitumor activity and hydrogen peroxide release by macrophages elicited by trehalose diesters. 680 86

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.
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PMID:Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR. 1245 66

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.
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PMID:The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis. 1631 72

We showed recently that actin assembly by phagosomal membranes facilitates fusion with late endocytic organelles in macrophages. Moreover, lipids that induced phagosomal actin also stimulated this fusion process. In macrophages infected with pathogenic mycobacteria actin-stimulatory lipids led to an increase in pathogen destruction, whereas inhibitors facilitated their growth. A model was proposed whereby phagosomal membrane actin assembly provides tracks for lysosomes to move towards phagosomes, thereby facilitating fusion. Here, we investigated how cAMP affected phagosomal actin assembly in vitro, and phagosomal actin, acidification and late fusion events in J774 macrophages. Latex bead phagosomes are shown to possess adenylyl cyclase activity, which synthesizes cAMP, and phosphodiesterase activity, which degrades cAMP. The system is regulated by protein kinase A (PKA). Increasing cAMP levels inhibited, whereas decreasing cAMP levels stimulated, actin assembly in vitro and within cells. Increasing cAMP levels also inhibited phagosome-lysosome fusion and acidification in cells, whereas reducing cAMP had the opposite effect. High cAMP levels induced an increase in intraphagosomal growth in macrophages of both the non-pathogenic Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis, whereas low cAMP levels or inhibition of PKA correlated with increased bacterial destruction. We argue that the phagosome cAMP-PKA system behaves as a molecular switch that regulates phagosome actin and maturation in macrophages.
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PMID:cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria. 1693 99

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.
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PMID:Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis. 1705 28

Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His(98)), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys(74)). We find that Rv0805 has a previously unappreciated 2',3'-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP. Changing His(98) to alanine or asparagine suppressed the 2',3'-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2',3'-cNMP phosphodiesterase by introducing histidine in lieu of Cys(74). YfcE-C74H cleaved the P-O2' bond of 2',3'-cAMP to yield 3'-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2' or P-O3' to yield a mixture of 3'-AMP and 2'-AMP products, with a bias toward 3'-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3' bond of 2',3'-cAMP to generate 2'-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.
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PMID:A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. 1875 71


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