Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026918 (Mycobacterium)
 52,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3',5'-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the DeltaMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the DeltaMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the DeltaMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.
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PMID:Cyclic AMP in mycobacteria: characterization and functional role of the Rv1647 ortholog in Mycobacterium smegmatis. 1839 Jun 60

Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 A. The structure of the apoenzyme reveals that the protein is composed of five alpha-helices with connecting loops and is a member of the alpha-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between alpha-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.
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PMID:The 1.25 A resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis. 1856 Jan 50

Mycobacterium tuberculosis adapts to cellular stresses such as decreased oxygen concentration, at least in part, by upregulation of the dormancy survival regulon, which is thought to be important for the bacterium's ability to enter a persistent state in its human host. We have determined the structure of hypoxic response protein 1, a protein encoded by one of the most strongly upregulated genes in the dormancy survival regulon. Hypoxic response protein 1 is an example of a 'cystathionine-beta-synthase-domain-only' protein; however, unlike other cystathionine-beta-synthase domains, it does not appear to bind AMP. The protein is proteolytically sensitive at its C-terminus and contains two unexpected disulfide bonds, one of which appears resistant to reducing agents in solution and is, therefore, most likely buried in the protein and is not solvent-accessible. We show that the protein is secreted from the bacterium in hypoxic in vitro culture and does not accumulate in the bacterial cell wall. The biological function of the protein remains unclear, but we suggest that it may contribute to the modulation of the host immune response. The work reported advances our understanding of the chemistry and cell biology of this intriguing and potentially important protein, and establishes a structural framework for future functional and immunological studies.
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PMID:The structure and unusual protein chemistry of hypoxic response protein 1, a latency antigen and highly expressed member of the DosR regulon in Mycobacterium tuberculosis. 1864 Jan 26

IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.
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PMID:CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen. 1864 43

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

Virulent Mycobacterium tuberculosis (Mtb) induces a maladaptive cytolytic death modality, necrosis, which is advantageous for the pathogen. We report that necrosis of macrophages infected with the virulent Mtb strains H37Rv and Erdmann depends on predominant LXA(4) production that is part of the antiinflammatory and inflammation-resolving action induced by Mtb. Infection of macrophages with the avirulent H37Ra triggers production of high levels of the prostanoid PGE(2), which promotes protection against mitochondrial inner membrane perturbation and necrosis. In contrast to H37Ra infection, PGE(2) production is significantly reduced in H37Rv-infected macrophages. PGE(2) acts by engaging the PGE(2) receptor EP2, which induces cyclic AMP production and protein kinase A activation. To verify a role for PGE(2) in control of bacterial growth, we show that infection of prostaglandin E synthase (PGES)(-/-) macrophages in vitro with H37Rv resulted in significantly higher bacterial burden compared with wild-type macrophages. More importantly, PGES(-/-) mice harbor significantly higher Mtb lung burden 5 wk after low-dose aerosol infection with virulent Mtb. These in vitro and in vivo data indicate that PGE(2) plays a critical role in inhibition of Mtb replication.
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PMID:Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death. 1895 68

5'-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS, 1) is a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase in Mycobacterium tuberculosis. This inhibitor acts by disrupting the biosynthesis of the mycobactin siderophores that are essential for the process of iron acquisition. To aid with in vitro metabolism and in vivo pharmacokinetic studies of Sal-AMS, a stable deuterium-labelled Sal-AMS analog (Sal-AMS-d(4)) was synthesized. This deuterium-labelled analog was used as an internal standard to conduct in vitro plasma and microsomal stability studies. Sal-AMS was found to be stable for 24 h in human plasma and 1 h in human liver microsomes at 37 degrees C.
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PMID:Synthesis of deuterium-labelled 5'-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS-d(4)) as an internal standard for quantitation of Sal-AMS. 1905 Jul 43

The gene for the Thermotoga maritima Trk potassium transporter component TrkA was originally thought to be a frameshift mutation and not to encode a functional protein. However, expression from this gene yielded a complex consisting of two distinct proteins designated TM1088A and -B. Genetic complementation of Escherichia coli mutants unable to transport potassium suggests that TM1088A/B is part of a functional Trk potassium transporter complex with the membrane protein TM1089. The protein structure for TM1088A shows a characteristic Rossmann fold indicating an NAD+ binding site and has structural similarity to potassium channel-related proteins. Ligand binding studies indicated that ATP, ADP, and AMP stabilized TM1088A to a much greater degree than NADH and NAD, consistent with the crystal structure of TM1088A, which contains a bound AMP natural ligand at the characteristic GXGXXG nucleotide binding site. Mutation of single and all glycines at this nucleotide binding site eliminated in vitro protein stabilization by the ligand, yet these mutated proteins could still functionally complement the E. coli potassium uptake mutants. We predict that this new two-subunit class of TrkA proteins is present in a number of organisms. A further subclass of the predicted two-subunit TrkA proteins lack an identifiable membrane-spanning subunit of the Trk K+ transporter. This class, as exemplified by Mycobacterium tuberculosis, did not complement E. coli potassium transport with the native E. coli TrkH; thus, it may require a novel TrkH-like protein for activity or provide an alternate function in vivo.
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PMID:The Thermotoga maritima Trk potassium transporter--from frameshift to function. 1916 17

The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty acid activation. These proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-CoA-synthesizing fatty acyl-CoA ligases (FACLs). Presently, it is not evident how obligate pathogens such as Mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyladenylates could indeed be a general mechanism. Here, based on elucidation of the first structure of an FAAL protein and by generating loss-of-function and gain-of-function mutants that interconvert FAAL and FACL activities, we demonstrate that an insertion motif dictates formation of acyladenylate. Because FAALs in Mtb are crucial nodes in the biosynthetic network of virulent lipids, inhibitors directed against these proteins provide a unique multipronged approach to simultaneously disrupting several pathways.
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PMID:Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis. 1918 84

Mycobacterium tuberculosis glutamyl-tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity. It was found to be similar to the other well-characterized GluRS, especially the E. coli enzyme, with respect to the requirement for bound tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.7 microm) and ATP (78 microm), but to differ by a one order of magnitude higher K(M) value for L-Glu (2.7 mm). At variance with the E. coli enzyme, among the several compounds tested as inhibitors, only pyrophosphate and the glutamyl-AMP analog glutamol-AMP were effective, with K(i) values in the mum range. The observed inhibition patterns are consistent with a random binding of ATP and L-Glu to the enzyme-tRNA complex. Mt-GluRS, which is predicted by genome analysis to be of the non-discriminating type, was not toxic when overproduced in E. coli cells indicating that it does not catalyse the mischarging of E. coli tRNA(Gln) with L-Glu and that GluRS/tRNA(Gln) recognition is species specific. Mt-GluRS was significantly more sensitive than the E. coli form to tryptic and chymotryptic limited proteolysis. For both enzymes chymotrypsin-sensitive sites were found in the predicted tRNA stem contact domain next to the ATP binding site. Mt-GluRS, but not Ec-GluRS, was fully protected from proteolysis by ATP and glutamol-AMP. Small-angle X-ray scattering showed that, at variance with the E. coli enzyme that is strictly monomeric, the Mt-GluRS monomer is present in solution in equilibrium with the homodimer. The monomer prevails at low protein concentrations and is stabilized by ATP but not by glutamol-AMP. Inspection of small-angle X-ray scattering-based models of Mt-GluRS reveals that both the monomer and the dimer are catalytically active. By using affinity chromatography and His(6)-tagged forms of either GluRS or glutamyl-tRNA reductase as the bait it was shown that the M. tuberculosis proteins can form a complex, which may control the flux of Glu-tRNA(Glu) toward protein or tetrapyrrole biosynthesis.
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PMID:Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl-tRNA synthetase and determination of its oligomeric structure in solution. 1918 40


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