Mechanism of Action of Showdomycin

Among the syntheses of DNA, RNA and protein in Escherichia coli cells, the DNA synthesis was found to be preferentially inhibited at lower concentrations of showdomycin. At such lower concentrations of this antibiotic, serious decreases in the synthesis of deoxycytidine phosphates and in de novo synthesis of deoxythymidine phosphates were found in parallel with the decrease in the synthesis of DNA, although the syntheses of other pyrimidine nucleotides were not significantly diminished. The salvage synthesis of deoxythymidine phosphates was very resistant to this antibiotic. The inhibitory action of this antibiotic on DNA synthesis could be reversed by the concomitant addition of a thiol compound or a nucleoside. When a nucleoside was added after the completion of the inhibition by showdomycin, the recovery of the DNA synthesis from the inhibition was detected only after the recovery of the syntheses of pyrimidine ribotides, pyrimidine deoxyribotides and RNA have become distinct.     

Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Mechanism of Action of Showdomycin

Showdomycin, 2-β-d-ribofuranosylmaleimide, inhibited the incorporation of amino acids and purine and pyrimidine bases into macromolecules in E. coli K-12 cells at low concentrations. The inhibitory action of showdomycin could be reversed by the addition of a nucleoside or a sulfhydryl compound. In marked contrast to common nucleosides, the pseudouridine showed no such effect. This may indicate that the N-glycosyl linkage in the nucleoside is a structural requirement for its reversing activity on the inhibitory action of showdomycin.

N-Ethylmaleimide, which has structural similarity to showdomycin, inhibited the incorporation of amino acids and purine and pyrimidine bases as well as showdomycin. The inhibitory action of N-ethylmaleimide, however, was not reversed by the addition of a nucleoside. This may indicate that there may be difference in the mechanism of the inhibitory action between N-ethylmaleimide and showdomycin.     

Formation of l-Butyl-5-hydroxy-methylpyrrole-2-carboxaldehyde in the Maillard Reaction between Lactose and n-Butylamine

A new mutant Shm^r-001-1 has been isolated by treating showdomycin-resistant mutant Shm^r-001 cells with N-methyl-N′-nitro-N-nitrosoguanidine. This mutant was resistant to high level of showdomycin, and took up practically no showdomycin and little pyrimidine nucleosides, and it showed different ability to take up purine nucleosides. Strains Shm^r-001–1, Shm^r-001, and K–12 (wild type) were compared: in susceptibility to showdomycin, in ability to take up the antibiotic and various nucleosides, on effects of other nucleosides on entry of particular nucleosides, and on kinetics of the entry of nucleosides and showdomycin. From these experiments, at least three different nucleoside transport systems were observed in Escherichia coli K–12 cells: the first system was common to adenine nucleosides, pyrimidine nucleosides, and showdomycin; the second system was common to adenine nucleosides, guanine nucleosides, inosine, pyrimidine nucleosides, and showdomycin; and the third system was common to adenine nucleosides, guanine nucleosides, and inosine. The first system was not observable in Shm^r-001 cells. In Shm^r-001–1 cells both the first and the second systems were no longer detectable but the third system was found to be active.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Microbial Transformation of Antibiotics

Microbial transformation of the nucleoside analogue antibiotic showdomycin was performed using some Streptomyces species. Both the growing culture and the resting cells of Streptomyces sp. No. 383 arrested the antibacterial activity of showdomycin. The inactivated showdomycin was isolated from the reaction mixture by carbon chromatography and was identified with an isoshowdomycin sample which has been chemically derived from showdomycin. It is conjectured that the conversion of showdomycin to isoshowdomycin results from isomerization by an enzyme of Streptomyces sp. No. 383.     

Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity

Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 A resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety.     

Thymidine-requiring mutants of Salmonella typhimurium that are defective in deoxyuridine 5''-phosphate synthesis.

In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.     

Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.

Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.     

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.     

Pyrimidine Nucleoside Salvage Confers an Advantage to Xenorhabdus nematophila in Its Host Interactions

Xenorhabdus nematophila is a mutualist of entomopathogenic nematodes and a pathogen of insects. To begin to examine the role of pyrimidine salvage in nutrient exchange between X. nematophila and its hosts, we identified and mutated an X. nematophila tdk homologue. X. nematophila tdk mutant strains had reduced virulence toward Manduca sexta insects and a competitive defect for nematode colonization in plate-based assays. Provision of a wild-type tdk allele in trans corrected the defects of the mutant strain. As in Escherichia coli, X. nematophila tdk encodes a deoxythymidine kinase, which converts salvaged deoxythymidine and deoxyuridine nucleosides to their respective nucleotide forms. Thus, nucleoside salvage may confer a competitive advantage to X. nematophila in the nematode intestine and be important for normal entomopathogenicity.     

Studies on the Biosynthesis of α-Putrescinylthymine in Bacteriophage φW-14-Infected Pseudomonas acidovorans

The alpha-putrescinylthymine (putThy) in bacteriophage phiW-14 DNA is synthesized at the mononucleotide level: it is labeled by uracil or deoxyuridine but not by thymidine, and it appears in the acid-soluble pool of infected cells before the onset of phage DNA synthesis. The methylene group at the C-5 position of the pyrimidine moiety of putThy is derived in vivo from a C(1) unit. Extracts of a phage infected thymidine auxotroph of the host, Pseudomonas acidovorans, apparently contain a phage-specific thymidylate synthetase and a phage-specific activity which forms 5-hydroxymethyl dUMP from N(5), N(10)-methylene-tetrahydrofolate and dUMP.     

Synthesis and Biological Activity of Various Selenenyl and Tellurenyl-Substituted Deoxyuridines and Deoxyuridylates

Abstract

5-Methylselenenyl and 5-phenyltellurenyldeoxyuridines were constructed via the transmetallation of the corresponding 5-bromo-3′,5′-bis(dimethyl-tert-butyl)silyl deoxyuridine with n-butyllithium followed by electrophilic trapping of the anion with the appropriate reagent. These substituted deoxyuridylates have been evaluated as potenital inhibitors of Lactobacillus casei thymidylate synthase.     

“Slim” Nucleosides and Nucleotides. I. Synthetic,Structural and Biophysical Investigations of Showdomycins

Abstract

A new, convenient, and short synthesis of 2′-deoxyshowdomycin, along with an improved procedure for the preparation of showdomycin, have been presented. A single-crystal X-ray structure of l-benzyl-2′-deoxyshowdomycin (9) has been reported. Conformational studies using C.D. indicated that showdomycin exists predominantly in an anti conformation in aqueous solution. Molecular mechanics calculations using AMBER point to comparable binding energy of showdomycin-adenosine pair with the natural uridine-adenosine pair, but with a significant base-ribose conformational deviation from the natural array in the former. Implications of such a conformational deviation on tumor and viral replications have been discussed. Base-pairing studies employing high resolution NMR spectroscopy indicate that both showdomycin and epishowdomycin base-pair with adenosine-5′-monophosphate (AMP); however, while showdomycin also shows evidence of stacking, that was absent in epishowdomycin. Molecular modeling studies using QUANTA/CHARMm show that showdomycin is capable of forming a homopolymer duplex by base-pairing with poly(A), but with a considerably broader and deeper major groove. A heteropolymer duplex with a single insert of showdomycin exhibits tighter coiling at the point of insertion. A ten-picosecond dynamics simulation of the above heteroduplex revealed relaxation of the helix with disruption of H- bonding for two base pairs on either side of the insertion point forming a large central cavity.     

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

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 CH₂H₄folate 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.     

Properties and inhibition of thymidylate synthetase in Drosophila melanogaster

A quantitative comparison of the incorporation of methyl-³H-thymidine and 6-³H-deoxyuridine into the DNA of Drosophila melanogaster in the presence and in the absence of 5-fluorouracil indicated that 5-fluorouracil inhibits the reaction converting dUMP to dTMP catalysed by thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, E.C. 2.1.1.b). The enzyme exhibits maximal activity at pH 7·5 to 8·0 and is protected from heat inactivation by deoxyuridine monophosphate. The addition of thiol compounds to the homogenization buffer results in the enhancement of synthetase activity. The K_m values for deoxyuridine monophosphate and 5,10-methylenetetrahydrofolate are 6·8 × 10^?6 M and 8·3 × 10^?5 M, respectively. Fluorodeoxyuridine monophosphate, trifluoromethyldeoxyuridine monophosphate, and methotrexate are inhibitors of the enzyme. 5-Bromodeoxyuridine and 5-iododeoxyuridine have no inhibitory effect. The results support the contention that, under conditions which induce morphological lesions in Drosophila, fluorinated pyrimidines and methotrexate inhibit the de novo synthesis of thymidylate whereas thymidine analogues function in some other manner.     

Showdomycin,a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase

Showdomycin [2-(β-d-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na⁺ + K⁺)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01·mol^?1·min^?1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K⁺, 100 μM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na⁺ + K⁺)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na⁺ + K⁺)-ATPase and inhibition is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.     

High-temperature solvolysis of 2′-deoxyribonucleosides in aqueous amine solutions

Abstract

Degradation of 2′-deoxyribonucleosides in 0.5?M aqueous pyrrolidine at 110?°C proceeds at different rates, ordered as deoxyuridine?>?deoxyadenosine?>?deoxycytidine?>?deoxyguanosine ??deoxythymidine. Deoxyadenosine degradation produces the free base, adenine, while deoxycytidine by deamination produces deoxyuridine, and then uracil. The solvolysis of deoxyadenosine has an activation energy of 23.3?kcal/mol. Ammonolysis is slower than pyrrolidinolysis for deoxyadenosine, but faster for deoxyguanosine. In pyrrolidinolysis of the trinucleotides, d-TGT and d-TAT, the guanine moiety reacts faster than the adenine moiety. These trends are interpreted in terms of the ionization of the guanine moieties under basic conditions, rendering them less susceptible to nucleophilic attack.     

Loop Stereochemistry and Dynamics in Transfer RNA

Abstract

The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology. Both loops are seven residues long and both present sharp turns after the second residue, U33 and ψ55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively. In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48. In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3′-end. At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule. The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet. Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed.