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)

The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site. The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5%. Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2%. The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions. A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis. These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state. We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop.
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PMID:Multicopy crystallographic refinement of a relaxed glutamine synthetase from Mycobacterium tuberculosis highlights flexible loops in the enzymatic mechanism and its regulation. 1214 52

The N-1-(5'-phosphoribosyl)-ATP transferase catalyzes the first step of the histidine biosynthetic pathway and is regulated by a feedback mechanism by the product histidine. The crystal structures of the N-1-(5'-phosphoribosyl)-ATP transferase from Mycobacterium tuberculosis in complex with inhibitor histidine and AMP has been determined to 1.8 A resolution and without ligands to 2.7 A resolution. The active enzyme exists primarily as a dimer, and the histidine-inhibited form is a hexamer. The structure represents a new fold for a phosphoribosyltransferase, consisting of three continuous domains. The inhibitor AMP binds in the active site cavity formed between the two catalytic domains. A model for the mechanism of allosteric inhibition has been derived from conformational differences between the AMP:His-bound and apo structures.
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PMID:Crystal structure of ATP phosphoribosyltransferase from Mycobacterium tuberculosis. 1251 75

Ak (adenylate kinase) is a ubiquitous enzyme that catalyses a reversible high-energy phosphoryl-transfer reaction between ATP and AMP to form ADP. In the present study, the Ak gene (adk) of Mycobacterium tuberculosis was cloned, expressed in Escherichia coli and purified as a glutathione S-transferase fusion protein. Purified Ak converted AMP into ADP in the presence of [gamma-32P]ATP or [gamma-32P]GTP. Replacement of arginine-88 of adk with glycine resulted in the loss of enzymic activity. The purified protein also showed Ndk (nucleoside diphosphate kinase)-like activity as it transferred terminal phosphate from [gamma-32P]ATP to all nucleoside diphosphates, converting them into corresponding triphosphates. However, Ndk-like activity of Ak was not observed with [gamma-32P]GTP. Immunoblot analysis of various cellular fractions of M. tuberculosis H37Rv revealed that Ak is a cytoplasmic protein. The dual activity of Ak as both nucleoside mono- and di-phosphate kinases suggested that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism.
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PMID:Nucleoside diphosphate kinase-like activity in adenylate kinase of Mycobacterium tuberculosis. 1279 61

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.
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PMID:Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis. 1459 27

Tuberculosis is the leading cause of death worldwide from a single infectious disease. Search of new therapeutic tools requires the discovery and biochemical characterization of new potential targets among the bacterial proteins essential for the survival and virulence. Among them are the nucleoside monophosphate kinases, involved in the nucleotide biosynthesis. In this work, we determined the solution structure of adenylate kinase (AK) from Mycobacterium tuberculosis (AKmt), a protein of 181 residues that was found to be essential for bacterial survival. The structure was calculated by a simulated annealing protocol and energy minimization using experimental restraints, collected by nuclear magnetic resonance spectroscopy. The final, well-defined 20 NMR structures show an average root-mean-square deviation of 0.77 A for the backbone atoms in regular secondary structure segments. The protein has a central CORE domain, composed of a five-stranded parallel beta-sheet surrounded by seven alpha-helices, and two peripheral domains, AMPbd and LID. As compared to other crystallographic structures of free form AKs, AKmt is more compact, with the AMP(bd) domain closer to the CORE of the protein. Analysis of the (15)N relaxation data enabled us to obtain the global rotational correlation time (9.19 ns) and the generalized order parameters (S(2)) of amide vectors along the polypeptide sequence. The protein exhibits restricted movements on a picosecond to nanosecond time scale in the secondary structural regions with amplitudes characterized by an average S(2)() value of 0.87. The loops beta1/alpha1, beta2/alpha2, alpha2/alpha3, alpha3/alpha4, alpha4/beta3, beta3/alpha5, alpha6/alpha7 (LID), alpha7/alpha8, and beta5/alpha9 exhibit rapid fluctuations with enhanced amplitudes. These structural and dynamic features of AKmt may be related to its low catalytic activity that is 10-fold lower than in their eukaryote counterparts.
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PMID:Structural and dynamic studies on ligand-free adenylate kinase from Mycobacterium tuberculosis revealed a closed conformation that can be related to the reduced catalytic activity. 1470 32

ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme of the histidine pathway, is a complex allosterically regulated enzyme, which controls the flow of intermediates through this biosynthetic pathway. The crystal structures of Escherichia coli ATP-PRT have been solved in complex with the inhibitor AMP at 2.7A and with product PR-ATP at 2.9A (the ribosyl-triphosphate could not be resolved). On the basis of binding of AMP and PR-ATP and comparison with type I PRTs, the PRPP and parts of the ATP-binding site are identified. These structures clearly identify the AMP as binding in the 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP)-binding site, with the adenosine ring occupying the ATP-binding site. Comparison with the recently solved Mycobacterium tuberculosis ATP-PRT structures indicates that histidine is solely responsible for the large conformational changes observed between the hexameric forms of the enzyme. The role of oligomerisation in inhibition and the structural basis for the synergistic inhibition by histidine and AMP are discussed.
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PMID:The structure of Escherichia coli ATP-phosphoribosyltransferase: identification of substrate binding sites and mode of AMP inhibition. 1474 Dec 9

The metabolic repertoire in nature is augmented by generating hybrid metabolites from a limited set of gene products. In mycobacteria, several unique complex lipids are produced by the combined action of fatty acid synthases and polyketide synthases (PKSs), although it is not clear how the covalently sequestered biosynthetic intermediates are transferred from one enzymatic complex to another. Here we show that some of the 36 annotated fadD genes, located adjacent to the PKS genes in the Mycobacterium tuberculosis genome, constitute a new class of long-chain fatty acyl-AMP ligases (FAALs). These proteins activate long-chain fatty acids as acyl-adenylates, which are then transferred to the multifunctional PKSs for further chain extension. This mode of activation and transfer of fatty acids is contrary to the previously described universal mechanism involving the formation of acyl-coenzyme A thioesters. Similar mechanisms may operate in the biosynthesis of other lipid-containing metabolites and could have implications in engineering novel hybrid products.
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PMID:Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. 1504 94

2-Methyladenosine (methyl-Ado) has selective activity against Mycobacterium tuberculosis (M. tuberculosis). In an effort to better understand its mechanism of action, we have characterized its metabolism in M. tuberculosis cells. The primary intracellular metabolite of methyl-Ado was 2-methyl-adenylate (methyl-AMP). Very little of the methyl-AMP was metabolized further. A M. tuberculosis strain that was resistant to methyl-Ado did not express adenosine kinase and did not convert methyl-Ado to methyl-AMP in intact cells. In contrast to these results, the primary intracellular metabolite of adenosine in M. tuberculosis cells was ATP, which was readily incorporated into RNA. The rate of metabolism of methyl-Ado to methyl-AMP was similar to the rate of metabolism of adenosine to ATP. Treatment of M. tuberculosis with methyl-Ado did not affect intracellular ATP levels. Methyl-Ado and Ado were also cleaved to 2-methyladenine and adenine, respectively, which accumulated in the medium outside the cells. These studies suggested that methyl-AMP was the active metabolite responsible for the cytotoxicity of this agent. Furthermore, because methyl-Ado was poorly metabolized in human cells, these studies indicated that the selective activity of methyl-Ado was due to its selective activation by M. tuberculosis. These studies have identified two enzyme reactions (Ado kinase and Ado cleavage) in M. tuberculosis that could be exploited for the rational design of new and selective anti-M. tuberculosis agents.
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PMID:Metabolism of 2-methyladenosine in Mycobacterium tuberculosis. 1520 8

Previous studies in our laboratory have shown a differential activation of the mitogen-activated protein kinases (MAPKs) in primary bone marrow-derived macrophages following infection with pathogenic Mycobacterium avium compared to the activation following infection with nonpathogenic Mycobacterium smegmatis. Additionally, M. smegmatis-infected macrophages produced significantly elevated levels of tumor necrosis factor alpha (TNF-alpha) compared to the levels produced by M. avium-infected macrophages. The TNF-alpha production was dependent on both p38 and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. However, the macrophage transcription factors downstream of the MAPKs, which were required for TNF-alpha production, remained undefined. In this study we determined that the transcription factor cyclic AMP response element binding protein (CREB) is significantly more activated in M. smegmatis-infected macrophages than in M. avium-infected macrophages. We also found that CREB activation was dependent on p38 and protein kinase A but not on ERK 1/2 or calmodulin kinase II. Moreover, mutating the cAMP-responsive element on the TNF-alpha promoter resulted in significantly diminished promoter activity following M. smegmatis infection but not M. avium infection. The inability of macrophages infected with M. avium to sustain MAPK activation and to produce high levels of TNF-alpha was due, in part, to an increase in serine/threonine phosphatase PP2A activity. Our studies are the first to demonstrate an important role for the transcription factor CREB in TNF-alpha production by mycobacterium-infected macrophages, as well as a role for M. avium's induction of PP2A phosphatase activity as a mechanism to limit macrophage activation.
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PMID:Differential activation of the transcription factor cyclic AMP response element binding protein (CREB) in macrophages following infection with pathogenic and nonpathogenic mycobacteria and role for CREB in tumor necrosis factor alpha production. 1561 91

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that cyclic AMP (cAMP) has an important, yet overlooked, role in mycobacteria. This study examined the effect of exogenous cAMP on protein expression in Mycobacterium bovis BCG grown under hypoxic versus ambient conditions. Both shaking and shallow standing cultures were examined for each atmospheric condition. Different cAMP-dependent changes in protein expression were observed in each condition by two-dimensional gel electrophoresis. Shaking low-oxygen cultures produced the most changes (12), while standing ambient conditions showed the fewest (2). Five upregulated proteins, Rv1265, Rv2971, GroEL2, PE_PGRS6a, and malate dehydrogenase, were identified from BCG by mass spectrometry and were shown to also be regulated by cAMP at the mRNA level in both M. tuberculosis H37Rv and BCG. To our knowledge, these data provide the first direct evidence for cAMP-mediated gene regulation in TB complex mycobacteria.
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PMID:Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions. 1580 14


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