Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0Å Resolution

Parthasarathy, S.; Turley, S.; Ulmer, Jonathan D.; Rhie, Ho Gun; Sibley, Carol Hopkins; Hól, Wim G. J.

doi:10.1016/j.jmb.2005.07.071

Cited by 70 publications

(160 citation statements)

References 33 publications

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“…The H69 residue has been suggested to function as a key catalytic residue and is highly conserved in all ThyX proteins, but its configuration varies in different structures. The H69 residue had previously been changed to glutamate to reverse the charge of the residue and failed to complement growth of the 2913 E. coli strain, which supports the catalytic role for this histidine residue (27). The residues H91 and E92 form one and two hydrogen bonds to dUMP, respectively.…”

Section: Complementationmentioning

confidence: 82%

“…We extended this analysis by testing residues within and surrounding the previously defined motif to identify amino acids required for full enzyme function and for oxidation and deprotonation activity in vitro. Since the structure of M. tuberculosis ThyX has been solved previously (27), we focused our attention on highly conserved amino acids and those shown to interact in crystals with the substrates and cofactors required for this complex reaction (Table 1). Figure 2 shows the alignment of this extended ThyX motif from several diverse eubacterial and archaeal species and highlights specific residues that are highly conserved.…”

Section: Resultsmentioning

confidence: 99%

“…thyX (Rv2754c) from M. tuberculosis H37Rv was cloned into a pUC18 vector for complementation analysis and into a pET24d vector with a six-His tag for high-level protein expression and purification as described previously (27). The cloned vectors are designated pUC18-ThyX and pET24d-ThyX, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The structures of the ThyX protein from Thermotoga maritima (16), M. tuberculosis (27), and Paramecium bursaria chlorella virus-1 (10) have been solved recently, and the basic structures are very similar. All three enzymes are homotetramers, and they are structurally and evolutionarily unrelated to any of the ThyA enzymes that have been studied (22,23).…”

mentioning

confidence: 99%

“…The genes that encode the putative ThyX enzyme have been identified in organisms from several hundred species, representing more than 70 genera, and there are some highly conserved amino acids that have been termed the ThyX motif (RHRX 7-8 S) (17,18). Moreover, a limited study of essential residues in the M. tuberculosis ThyX enzyme confirmed that they were conserved in the orthologous H. pylori enzyme (17,27).…”

mentioning

confidence: 99%

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Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

et al. 2008

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A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX 7-8 S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ⌬thyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X 24 H69X 25 R95H RX 7 S105XRYX 90 R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

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Section: Complementationmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

et al. 2008

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Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases, and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

Kögler

Busson

Jonghe

et al. 2012

Chemistry & Biodiversity

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A series of 5-substituted analogs of 6-aza-2'-deoxyuridine 5'-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt.

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Synthesis, Biological and Molecular Docking Studies of Thiazole‐Thiadiazole derivatives as potential Anti‐Tuberculosis Agents

Shaikh,

Labhade,

Kale

et al. 2024

Chemistry & Biodiversity

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Tuberculosis remains a global health threat, with increasing infection rates and mortality despite existing anti‐TB drugs. The present work focuses on the research findings regarding the development and evaluation of thiadiazole‐linked thiazole derivatives as potential anti‐tuberculosis agents. We present the synthesis data and confirm the compound structures using spectroscopic techniques. The current study reports twelve thiazole‐thiadiazole compounds (5a‐5l) for their anti‐tuberculosis and related bioactivities. This paper emphasizes compounds 5g, 5i, and 5l, which exhibited promising MIC values, leading to further in silico and interaction analysis. Pharmacophore mapping data included in the present analysis identified tubercular ThyX as potential drug targets. The compounds were evaluated for anti‐tubercular activity using standard methods, revealing significant MIC values, particularly compound 5l, with the best MIC value of 7.1285 µg/ml. Compounds 5g and 5i also demonstrated moderate to good MIC values against M. tuberculosis (H37Ra). Structural inspection of the docked poses revealed interactions such as hydrogen bonds, halogen bonds, and interactions containing Pi electron cloud, shedding light on conserved interactions with residues like Arg 95, Cys 43, His 69, and Arg 87 from the tubercular ThyX enzyme.

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Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0Å Resolution

Cited by 70 publications

References 33 publications

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases, and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

Synthesis, Biological and Molecular Docking Studies of Thiazole‐Thiadiazole derivatives as potential Anti‐Tuberculosis Agents

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