flavin-adenine-dinucleotide and 2--deoxyuridylic-acid

The bacterial thymidylate synthase ThyX is a multisubstrate flavoenzyme that takes part in the de novo synthesis of thymidylate in a variety of microorganisms. Herein we study the effect of FAD and dUMP binding on the thermal stability of wild type (WT) ThyX from the mesophilic Paramecium bursaria chlorella virus-1 (PBCV-1) and from the thermophilic bacterium Thermotoga maritima (TmThyX), and from two variants of TmThyX, Y91F and S88W, using differential scanning calorimetry. The energetics underlying these processes was characterized by isothermal titration calorimetry. The PBCV-1 protein is significantly less stable against the thermal challenge than the TmThyX WT. FAD exerted stabilizing effect greater for PBCV-1 than for TmThyX and for both mutants, whereas binding of dUMP to FAD-loaded proteins stabilized further only TmThyX. Different thermodynamic signatures describe the FAD binding to the WT ThyX proteins. While TmThyX binds FAD with a low μM binding affinity in a process characterized by a favorable entropy change, the assembly of PBCV-1 with FAD is governed by a large enthalpy change opposed by an unfavorable entropy change resulting in a relatively strong nM binding. An enthalpy-driven formation of a high affinity ternary ThyX/FAD/dUMP complex was observed only for TmThyX.

Topics: Calorimetry; Calorimetry, Differential Scanning; Coenzymes; Deoxyuracil Nucleotides; Enzyme Stability; Flavin-Adenine Dinucleotide; Mutant Proteins; Protein Binding; Substrate Specificity; Temperature; Thermotoga maritima; Thymidylate Synthase; Transition Temperature

ThyX, a flavin-dependent thymidylate synthase that is involved in the synthesis of dTMP from dUMP, is a promising target for the development of novel antibacterial drugs that aimed at blocking the biosynthesis of dTMP, one of the building blocks of DNA. This enzyme has been recently identified in some dsDNA viruses and pathogenic bacteria, including the gastric pathogen Helicobacter pylori. It shares neither sequence nor structural homology with the classical ThyA in humans and other organisms. Further more, ThyX and ThyA are the only source of dTMP in these organisms and other pathways cannot substitute for their function. Thus, ThyX-specific inhibitors could be effective antibacterial reagents while having no impact on human cells. Here we report the crystal structure of ThyX from Helicobacter pylori strain 26695 in complex with co-factor FAD and substrate dUMP at 2.5 A resolution, which consists of a 1.5 tetramer of ThyX with a total of 1248 residues, six FAD and six dUMP molecules in an asymmetric unit. The structure revealed the key residues that are involved in co-factor FAD and substrate dUMP binding, site-directed mutagenesis were performed to analysis the importance of these residues on ThyX activity by genetic complementation and FAD binding assay.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Genetic Complementation Test; Helicobacter pylori; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Sequence Alignment; Thymidylate Synthase

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K(m) values of the two substrates dUMP and CH(2) H(4) folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.

Topics: Amino Acid Sequence; Binding Sites; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Helicobacter pylori; Kinetics; Models, Molecular; Molecular Sequence Data; NADP; Recombinant Proteins; Sequence Alignment; Tetrahydrofolates; Thymidylate Synthase

Topics: Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Flavins; Helicobacter pylori; Humans; Thermotoga maritima; Thymidine Monophosphate; Thymidylate Synthase; Uracil

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2'-deoxyuridine-5'-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2'-deoxyuridine-5'-monophosphate. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (that is, a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2'-deoxythymidine-5'-monophosphate. These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

Topics: Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Deoxyuracil Nucleotides; Deuterium; Electrons; Flavin-Adenine Dinucleotide; Flavins; Helicobacter pylori; Humans; Magnetic Resonance Spectroscopy; Methylation; Models, Molecular; Mycobacterium tuberculosis; Protons; Thermotoga maritima; Thymidine; Thymidine Monophosphate; Thymidylate Synthase; Uracil

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 delta 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)R95HRX(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.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Bacterial Proteins; Deoxyuracil Nucleotides; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Flavin-Adenine Dinucleotide; Genetic Complementation Test; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutation; Mycobacterium tuberculosis; NADP; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Thymidine; Thymidylate Synthase

An unexpected substrate-dependent lag-phase, found in the single turnover reduction of FDTS bound flavin, sheds light on the molecular mechanism of this alternative thymidylate synthase.

Topics: Bacterial Proteins; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; NADP; Thermotoga maritima; Thymidylate Synthase

The synthesis of thymine for DNA is catalyzed by the enzyme thymidylate synthase (TS). A family of flavin-dependent TSs encoded by the thyX gene has been discovered recently. These newly discovered TSs require a reducing substrate in addition to 2'-deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (CH2THF), suggesting that the enzyme-bound flavin is a redox intermediary in catalysis. The oxidation of the reduced flavin of the TS from Campylobacter jejuni has been observed directly upon mixing with dUMP and CH2THF under anaerobic conditions, thus providing the first direct demonstration of its redox role in catalysis. Product analysis showed that the one mole of 2'-deoxythymidine monophosphate is formed along with one mole of tetrahydrofolate for each mole of reduced enzyme-bound flavin. The classic TS inactivator 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) was able to bind to the reduced enzyme but was unable to oxidize the flavin, even in the presence of CH2THF. Furthermore, the nucleotide binding site of the enzyme treated with FdUMP and CH2THF was irreversibly blocked, suggesting the formation of a stable substrate adduct analogous to that formed by the well-studied thyA-encoded TS. The formation of inactivated enzyme without flavin oxidation indicates that methylene transfer from the folate to the nucleotide occurs prior to flavin redox chemistry.

Topics: Campylobacter jejuni; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Oxidation-Reduction; Spectrophotometry; Tetrahydrofolates; Thymidine Monophosphate; Thymidylate Synthase

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M.tuberculosis ThyX (MtbThyX) enzyme complements the E.coli chi2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

Topics: Amino Acid Sequence; Binding Sites; Coenzymes; Crystallography, X-Ray; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Flavins; Molecular Sequence Data; Mycobacterium tuberculosis; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment; Thermotoga maritima; Thymidylate Synthase

Sequence analysis of the 330-kb double-stranded DNA genome of Paramecium bursaria chlorella virus-1 revealed an open reading frame A674R that encodes a protein with up to 53% amino acid identity to a recently discovered new class of thymidylate synthases, called ThyX. Unlike the traditional thymidylate synthase, ThyA, that uses methylenetetrahydrofolate (CH(2)H(4)folate) as both a source of the methylene group and the reductant, CH(2)H(4)folate only supplies the methylene group in ThyX-catalyzed reactions. Furthermore, ThyX only catalyzes thymidylate (dTMP) formation in the presence of reduced pyridine nucleotides and oxidized FAD. The distribution and transcription patterns of the a674r gene in Chlorella viruses were examined. The a674r gene was cloned, and the protein was expressed in Escherichia coli. Biochemical characterization of the P. bursaria chlorella virus-1 recombinant ThyX protein indicates that it is more efficient at converting dUMP to dTMP than previously studied ThyX enzymes, thus allowing more detailed mechanistic studies of the enzyme. The ThyX-dUMP complexes with bound FAD function as efficient NAD(P)H oxidases, indicating that dUMP binds to the enzyme prior to NAD(P)H. This oxidation activity is directly linked to FAD reduction. Our results indicate that ThyX-specific inhibitors can be designed that do not affect ThyA enzymes. Finally, a model is proposed for the early stages of ThyX catalysis.

Topics: Animals; Chlorella; Cloning, Molecular; Deoxyuracil Nucleotides; DNA, Viral; Enzyme Inhibitors; Escherichia coli; Flavin-Adenine Dinucleotide; Gene Expression; Nucleic Acid Hybridization; Oxidation-Reduction; Paramecium; Phycodnaviridae; RNA; Thymidine Monophosphate; Thymidylate Synthase; Transcription, Genetic; Virus Replication

The structure of thymidylate synthase complementing protein with substrates dUMP and FAD, presented in this issue of Structure, sheds light on a fascinating new catalytic mechanism, suggests a strategy for the design of new antimicrobial compounds, and highlights the promise of proteomics in medicine.

Topics: Deoxyuracil Nucleotides; Drug Design; Flavin-Adenine Dinucleotide; Humans; Molecular Structure; Thymidylate Synthase

Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor. The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor. We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family. The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Infections; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Folding; Protein Structure, Tertiary; Thermotoga maritima; Thymidylate Synthase

Although deoxythymidylate cannot be provided directly by ribonucleotide reductase, the gene encoding thymidylate synthase ThyA is absent from the genomes of a large number of nonsymbiotic microbes. We show that ThyX (Thy1) proteins of previously unknown function form a large and distinct class of thymidylate synthases. ThyX has a wide but sporadic phylogenetic distribution, almost exclusively limited to microbial genomes lacking thyA. ThyX and ThyA use different reductive mechanisms, because ThyX activity is dependent on reduced flavin nucleotides. Our findings reveal complexity in the evolution of thymidine in present-day DNA. Because ThyX proteins are found in many pathogenic microbes, they present a previously uncharacterized target for antimicrobial compounds.

Topics: Catalysis; Deoxyuracil Nucleotides; Electron Transport; Escherichia coli; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Gene Transfer, Horizontal; Genes, Archaeal; Genes, Bacterial; Helicobacter pylori; Molecular Weight; Phylogeny; Pyrococcus; Recombinant Proteins; Tetrahydrofolates; Thymidine Monophosphate; Thymidylate Synthase; Transformation, Bacterial; Uridine Monophosphate

Topics: Bacteria; Binding Sites; Crystallography, X-Ray; Deoxyuracil Nucleotides; Drug Design; Enzyme Inhibitors; Evolution, Molecular; Flavin-Adenine Dinucleotide; Helicobacter pylori; Humans; Methyltransferases; Models, Molecular; Phylogeny; Protein Conformation; Protein Structure, Tertiary; Protozoan Proteins; Tetrahydrofolates; Thermotoga maritima; Thymidine Monophosphate; Thymidylate Synthase