ABSTRACT
Deoxythymidine diphosphate (dTDP)-activated sugar nucleotides are the most diverse sugar nucleotides in nature. They serve as the glycosylation donors of glycosyltransferases to produce various carbohydrate structures in living organisms. However, most of the dTDP-sugars are difficult to obtain due to synthetic difficulties. The limited availability of dTDP-sugars has hindered progress in investigating the biosynthesis of carbohydrates and exploring new glycosyltransferases in nature. In this study, based on the de novo and salvage biosynthetic pathways, a variety of dTDP-activated sugar nucleotides were successfully prepared in high yields and on a large scale from readily available starting materials. The produced sugar nucleotides could provide effective tools for fundamental research in glycoscience.
Subject(s)
Nucleotides , Thymine Nucleotides , Thymine Nucleotides/chemistry , Nucleotides/metabolism , Glycosylation , Glycosyltransferases/metabolism , HexosesABSTRACT
N-acetylated sugars are often found, for example, on the lipopolysaccharides of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N-acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919-RS02330 putatively encoding an N-acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP-3-amino-3,6-dideoxy-d-glucose as a substrate as well as dTDP-3-amino-3,6-dideoxy-d-galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X-ray crystallographic structures of various complexes of the H. pullorum wild-type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N-acetyltransferase places it into the Class II left-handed-ß-helix superfamily (LßH). Taken together, the data presented herein suggest that 3-acetamido-3,6-dideoxy-d-glucose (or the galactose derivative) is found on either the H. pullorum O-antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non-pylori Helicobacter spp. have genes encoding N-acetyltransferases. Given that there is little information concerning the complex glycans in non-pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens.
Subject(s)
Acetyltransferases/chemistry , Bacterial Proteins/chemistry , Deoxy Sugars/chemistry , Helicobacter/enzymology , Lipopolysaccharides/chemistry , Thymine Nucleotides/chemistry , Acetyltransferases/genetics , Acetyltransferases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Deoxy Sugars/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Glycoconjugates/chemistry , Glycoconjugates/metabolism , Helicobacter/chemistry , Kinetics , Lipopolysaccharides/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thymine Nucleotides/metabolismABSTRACT
Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. We demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.
Subject(s)
DNA/chemistry , Nanostructures/chemistry , Polynucleotides/chemistry , DNA Nucleotidylexotransferase/chemistry , Deoxyuracil Nucleotides/chemistry , Nucleic Acid Conformation , Polymerization , Proof of Concept Study , Thymine Nucleotides/chemistryABSTRACT
PLD3 and PLD4 have recently been revealed to be endosomal exonucleases that regulate the innate immune response by digesting the ligands of nucleic acid sensors. These enzymes can suppress RNA and DNA innate immune sensors like toll-like receptor 9, and PLD4-deficent mice exhibit inflammatory disease. Targeting these immunoregulatory enzymes presents an opportunity to indirectly regulate innate immune nucleic acid sensors that could yield immunotherapies, adjuvants, and nucleic acid drug stabilizers. To aid in delineating the therapeutic potential of these targets, we have developed a high-throughput fluorescence enzymatic assay to identify modulators of PLD3 and PLD4. Screening of a diversity library (N = 17952) yielded preferential inhibitors of PLD3 and PLD4 in addition to a PLD3 selective activator. The modulation models of these compounds were delineated by kinetic analysis. This work presents an inexpensive and simple method to identify modulators of these immunoregulatory exonucleases.
Subject(s)
Enzyme Activators/chemistry , Enzyme Inhibitors/chemistry , Exodeoxyribonucleases/antagonists & inhibitors , Phospholipase D/antagonists & inhibitors , Enzyme Assays , Fluorescent Dyes/chemistry , High-Throughput Screening Assays , Humans , Nitrophenols/chemistry , Thymine Nucleotides/chemistry , Umbelliferones/chemistryABSTRACT
Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).
Subject(s)
Cross-Linking Reagents/chemistry , DNA/chemistry , Ketones/chemistry , Peptides/chemistry , Proteins/chemistry , Thymine Nucleotides/chemistry , Animals , Arginine/chemistry , Cattle , Cross-Linking Reagents/chemical synthesis , DNA/chemical synthesis , Histones/chemistry , Ketones/chemical synthesis , Serum Albumin, Bovine/chemistry , Thymine Nucleotides/chemical synthesis , Tumor Suppressor Protein p53/chemistryABSTRACT
The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.
Subject(s)
DNA Polymerase III/chemistry , DNA Polymerase III/metabolism , DNA/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Cryoelectron Microscopy , DNA/chemistry , DNA Polymerase III/ultrastructure , Dideoxynucleotides/chemistry , Dideoxynucleotides/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Models, Molecular , Mutation/genetics , Neoplasms/genetics , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , Thymine Nucleotides/chemistry , Thymine Nucleotides/metabolismABSTRACT
Triazoloacridinone C-1305, a potent antitumor agent recommended for Phase I clinical trials, exhibits high activity towards a wide range of experimental colon carcinomas, in many cases associated with complete tumor regression. C-1305 is a well-established dsDNA intercalator, yet no information on its mode of binding into DNA is available to date. Herein, we present the NMR-driven and MD-refined reconstruction of the 3D structures of the d(CGATATCG)2:C-1305 and d(CCCTAGGG)2:C-1305 non-covalent adducts. In both cases, the ligand intercalates at the TA/TA site, forming well-defined dsDNA:drug 1:1 mol/mol complexes. Orientation of the ligand within the binding site was unambiguously established by the DNA/ligand proton-proton NOE contacts. A subsequent, NMR-driven study of the sequence-specificity of C-1305 using a series of DNA duplexes, allowed us to confirm a strong preference towards TA/TA dinucleotide steps, followed by the TG/CA steps. Interestingly, no interaction at all was observed with duplexes containing exclusively the AT/AT, GG/CC and GA/TC steps.
Subject(s)
Acridines/chemistry , Adenine Nucleotides/chemistry , Antineoplastic Agents/chemistry , DNA/chemistry , Drug Discovery/methods , Intercalating Agents/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Thymine Nucleotides/chemistry , Triazoles/chemistry , Base Sequence , Binding Sites , Ligands , Models, Molecular , Molecular Dynamics Simulation , Nucleic Acid ConformationABSTRACT
The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.
Subject(s)
Click Chemistry/methods , Copper/chemistry , Deoxyribonucleotides/analysis , Deoxyuracil Nucleotides/chemistry , Cycloaddition Reaction , Deoxyadenine Nucleotides/analysis , Deoxyadenine Nucleotides/chemistry , Deoxycytosine Nucleotides/analysis , Deoxycytosine Nucleotides/chemistry , Deoxyguanine Nucleotides/analysis , Deoxyguanine Nucleotides/chemistry , Deoxyribonucleotides/chemistry , HCT116 Cells , HEK293 Cells , Humans , K562 Cells , Rhodamines/chemistry , Staining and Labeling , Thymine Nucleotides/analysis , Thymine Nucleotides/chemistryABSTRACT
Nucleosides analogues are the cornerstone of the treatment of several human diseases. They are especially at the forefront of antiviral therapy. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphate form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. In this context, it is mandatory to develop a rapid, reliable and sensitive enzyme activity test to evaluate their metabolic pathways. In this study, we report a proof of concept to directly monitor on-line nucleotide multiple phosphorylation. The methodology was developed by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry detection. Human Thymidylate Kinase (hTMPK) and human Nucleoside Diphosphate Kinase (hNDPK) were covalently immobilized on functionalized silica beads, and packed into micro-bioreactors (40⯵L). By continuous infusion of substrate into the bioreactors, the conversion of thymidine monophosphate (dTMP) into its di- (dTDP) and tri-phosphorylated (dTTP) forms was visualized by monitoring their Extracted Ion Chromatogram (EIC) of their [Mâ¯-â¯H]- ions. Both bioreactors were found to be robust and durable over 60 days (storage at 4⯰C in ammonium acetate buffer), after 20 uses and more than 750â¯min of reaction, making them suitable for routine analysis. Each on-line conversion step was shown rapid (<5â¯min), efficient (conversion efficiencyâ¯>â¯55%), precise and repeatable (CVâ¯<â¯3% for run-to-run analysis). The feasibility of the on-line multi-step conversion from dTMP to dTTP was also proved. In the context of selective antiviral therapy, this proof of concept was then applied to the monitoring of specificity of conversion of two synthesized Acyclic Nucleosides Phosphonates (ANPs), regarding human Thymidylate Kinase (hTMPK) and vaccina virus Thymidylate Kinase (vvTMPK).
Subject(s)
Bioreactors , Enzymes, Immobilized/chemistry , Nucleoside-Phosphate Kinase/chemistry , Organophosphonates/chemistry , Thymidine Monophosphate/chemistry , Thymine Nucleotides/chemistry , Humans , Mass Spectrometry/methods , Phosphorylation , Proof of Concept Study , Vaccinia virus/enzymologyABSTRACT
Proteins forming dimers or larger complexes can be strongly influenced by their effector-binding status. We investigated how the effector-binding event is coupled with interface formation via computer simulations, and we quantified the correlation of two types of contact interactions: between the effector and its binding pocket and between protein monomers. This was achieved by connecting the protein dynamics at the monomeric level with the oligomer interface information. We applied this method to ribonucleotide reductase (RNR), an essential enzyme for de novo DNA synthesis. RNR contains two important allosteric sites, the s-site (specificity site) and the a-site (activity site), which bind different effectors. We studied these different binding states with atomistic simulation and used their coarse-grained contact information to analyze the protein dynamics. The results reveal that the effector-protein dynamics at the s-site and dimer interface formation are positively coupled. We further quantify the resonance level between these two events, which can be applied to other similar systems. At the a-site, different effector-binding states (ATP vs dATP) drastically alter the protein dynamics and affect the activity of the enzyme. On the basis of these results, we propose a new mechanism of how the a-site regulates enzyme activation.
Subject(s)
Ribonucleotide Reductases/metabolism , Thymine Nucleotides/metabolism , Allosteric Regulation/physiology , Allosteric Site , Catalytic Domain , Humans , Molecular Dynamics Simulation , Protein Multimerization/physiology , Ribonucleotide Reductases/chemistry , Thymine Nucleotides/chemistryABSTRACT
Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2 H-pyridazin-6-yl (PzO)-a uracil analogue lacking a 2-keto group-as the nucleobase. Theoretical analyses and hybridization experiments indicated that PzO recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside 5'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside 5'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3'-nucleotide residue harboring PzO as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the PzO base at the 3' end could function in subsequent chain elongation. These properties of PzO were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.
Subject(s)
DNA Primers/genetics , Pyridazines/chemistry , Thymine Nucleotides/chemistry , Base Pairing , DNA Primers/metabolism , Electrons , Hydrogen Bonding , Models, Molecular , Static Electricity , Thymine Nucleotides/metabolismABSTRACT
Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delivery applications and gave fundamental data on their size distribution and stability. Graphical abstract á .
Subject(s)
Anti-HIV Agents/administration & dosage , Coordination Complexes/chemistry , Drug Carriers/chemistry , Metal-Organic Frameworks/chemistry , Nanoparticles/chemistry , Zidovudine/administration & dosage , Anti-HIV Agents/chemistry , Antimetabolites/administration & dosage , Antimetabolites/chemistry , Dideoxynucleotides/administration & dosage , Dideoxynucleotides/chemistry , Dynamic Light Scattering , Fractionation, Field Flow , Models, Molecular , Particle Size , Refractometry , Spectrophotometry, Ultraviolet , Thymine Nucleotides/administration & dosage , Thymine Nucleotides/chemistry , Zidovudine/analogs & derivatives , Zidovudine/chemistryABSTRACT
Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.
Subject(s)
Antibodies/chemistry , DNA, Single-Stranded/chemistry , Methyltransferases/analysis , Selenium/chemistry , Thymine Nucleotides/chemistry , Antibodies/metabolism , DNA, Single-Stranded/metabolism , Humans , Methyltransferases/metabolism , Selenium/metabolism , Thymine Nucleotides/metabolismABSTRACT
DesII is a radical SAM lyase that catalyzes a deamination reaction during the biosynthesis of desosamine in Streptomyces venezuelae. Competing mechanistic hypotheses for this radical-mediated reaction are differentiated according to whether a 1,2-migration takes place and the timing of proton abstraction following generation of a substrate α-hydroxyalkyl radical intermediate. In this study, the deuterated C4 epimer of the natural substrate, TDP-4-amino-4-deoxy-d-[3-2H]fucose, was prepared and shown to be a substrate for DesII undergoing deamination alone with a specific activity that is only marginally reduced (â¼3-fold) with respect to that of deamination of the natural substrate. Furthermore, pH titration of the deamination reaction implicates the presence of a hydron acceptor that facilitates catalysis but does not appear to be necessary. On the basis of these as well as previously reported results, a mechanism involving direct elimination of ammonium with concerted proton transfer to the nucleofuge from the adjacent α-hydroxyalkyl radical is proposed.
Subject(s)
Fucose/chemistry , Nucleoside Diphosphate Sugars/chemistry , Amino Sugars , Ammonium Compounds/metabolism , Catalysis , Deamination , Fucose/metabolism , Nucleoside Diphosphate Sugars/metabolism , Oxidoreductases/metabolism , S-Adenosylmethionine/metabolism , Streptomyces/enzymology , Thymine Nucleotides/chemistryABSTRACT
Many bacteria require l-rhamnose as a key cell wall component. This sugar is transferred to the cell wall using an activated donor dTDP-l-rhamnose, which is produced by the dTDP-l-rhamnose biosynthetic pathway. We determined the crystal structure of the second enzyme of this pathway dTDP-α-d-glucose 4,6-dehydratase (RfbB) from Bacillus anthracis. Interestingly, RfbB only crystallized in the presence of the third enzyme of the pathway RfbC; however, RfbC was not present in the crystal. Our work represents the first complete structural characterization of the four proteins of this pathway in a single Gram-positive bacterium.
Subject(s)
Bacillus anthracis/enzymology , Hydro-Lyases/chemistry , Nucleoside Diphosphate Sugars/chemistry , Protein Conformation , Thymine Nucleotides/chemistry , Bacillus anthracis/pathogenicity , Biosynthetic Pathways/genetics , Carbohydrate Epimerases/chemistry , Crystallography, X-RayABSTRACT
We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate (pTFT). The conjugate allows for direct optical and 19F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.
Subject(s)
Antineoplastic Agents/pharmacology , Nucleotides/chemistry , Serum Albumin/chemistry , Thymine Nucleotides/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Hydrogen-Ion Concentration , Molecular Structure , Oxidation-Reduction , Structure-Activity Relationship , Thymine Nucleotides/chemistryABSTRACT
The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose into dTDP-4-formamido-4,6-dideoxyglucose using N10-formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 104 M-1 s-1. In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N5-formyltetrahydrofolate and dTDP-4-amino-4,6-dideoxyglucose. The final model of Rv3404c was refined to an overall R-factor of 16.8% at 1.6 Å resolution. The results described herein are especially intriguing given that there have been no published reports of N-formylated sugars associated with M. tuberculosis. The data thus provide a new avenue of research into this fascinating, yet deadly, organism that apparently has been associated with human infection since ancient times.
Subject(s)
Bacterial Proteins/chemistry , Hydroxymethyl and Formyl Transferases/chemistry , Models, Molecular , Mycobacterium tuberculosis/enzymology , Virulence Factors/chemistry , Bacterial Proteins/metabolism , Catalysis , Crystallography, X-Ray , Deoxy Sugars/chemistry , Deoxy Sugars/metabolism , Formyltetrahydrofolates/chemistry , Formyltetrahydrofolates/metabolism , Hydroxymethyl and Formyl Transferases/metabolism , Kinetics , Mycobacterium tuberculosis/pathogenicity , Thymine Nucleotides/chemistry , Thymine Nucleotides/metabolism , Virulence Factors/metabolismABSTRACT
N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a "foothold" and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/biosynthesis , Deoxyadenosines/metabolism , Base Pairing , Catalytic Domain , Crystallography, X-Ray , DNA/chemistry , DNA-Directed DNA Polymerase/chemistry , Deoxyadenosines/chemistry , Deoxycytosine Nucleotides/chemistry , Deoxycytosine Nucleotides/metabolism , Humans , Kinetics , Protein Structure, Quaternary , Thymine Nucleotides/chemistry , Thymine Nucleotides/metabolism , DNA Polymerase iotaABSTRACT
DNA can be damaged by many compounds in our environment, and the resulting damaged DNA is commonly replicated by translesion synthesis (TLS) polymerases. Because the mechanism and efficiency of TLS are affected by the type of DNA damage, obtaining information for a variety of DNA adducts is critical. However, there is no structural information for the insertion of a dNTP opposite an O6-dG adduct, which is a particularly harmful class of DNA lesions. We used molecular dynamics (MD) simulations to investigate structural and energetic parameters that dictate preferred dNTP insertion opposite O6-benzyl-guanine (Bz-dG) by DNA polymerase IV, a prototypical TLS polymerase. Specifically, MD simulations were completed on all possible ternary insertion complexes and ternary -1 base deletion complexes with different Bz-dG conformations. Our data suggests that the purines are unlikely to be inserted opposite anti- or syn-Bz-dG, and dTTP is unlikely to be inserted opposite syn-Bz-dG, because of changes in the active site conformation, including critical hydrogen-bonding interactions and/or reaction-ready parameters compared to natural dG replication. In contrast, a preserved active site conformation suggests that dCTP can be inserted opposite either anti- or syn-Bz-dG and dTTP can be inserted opposite anti-Bz-dG. This is the first structural explanation for the experimentally observed preferential insertion of dCTP and misincorporation of dTTP opposite Bz-dG. Furthermore, we provide atomic level insight into why Bz-dG replication does not lead to deletion mutations, which is in contrast with the replication outcomes of other adducts. These findings provide a basis for understanding the replication of related O6-dG adducts.
Subject(s)
Benzyl Compounds/chemical synthesis , DNA Adducts/chemistry , DNA Polymerase beta/chemistry , DNA Repair , DNA Replication , Deoxyguanine Nucleotides/chemistry , Escherichia coli Proteins/chemistry , Guanine/chemical synthesis , Catalytic Domain , DNA Damage , DNA Polymerase beta/genetics , DNA Polymerase beta/metabolism , Deoxyadenine Nucleotides/chemistry , Deoxyadenine Nucleotides/metabolism , Deoxycytosine Nucleotides/chemistry , Deoxycytosine Nucleotides/metabolism , Deoxyguanine Nucleotides/metabolism , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Guanine/analogs & derivatives , Hydrogen Bonding , Molecular Dynamics Simulation , Mutagenesis , Protein Structure, Secondary , Protein Structure, Tertiary , Thymine Nucleotides/chemistry , Thymine Nucleotides/metabolismABSTRACT
The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.