Investigation of tautomeric composition of cytosine by multiphoton excitation spectra. I. Resonance hyper-Raman spectra

For the first time the quantum-mechanical calculations of intensity distribution in the resonance hyper-Raman spectra of the six tautomeric forms of cytosine were performed. It has been confirmed that in the aqueous solution of cytosine (pH 3.0) there exist several molecular structures: cytosine in the canonical amino-oxo tautomeric form, cation, as well as cys-imino-oxo and cys-amino-hydroxy tautomers; in the gaseous phase at 235°C cys-imino-oxotautomer dominates with the presence of small amount of cys-, trans-, amino-hydroxy tautomers and the caconical form of cytosine.     

Misincorporation of 2'-deoxyoxanosine into DNA: a molecular basis for NO-induced mutagenesis derived from theoretical calculations

A wide range of theoretical methods, including high level ab initio, density functional, self-consistent reaction field, molecular dynamics and thermodynamic integration calculations, have been used to analyze the mutagenic properties of oxanosine. The major tautomeric forms in the gas phase and aqueous solution have been determined. The ability of oxanosine to recognize thymine and cytosine in the gas phase and in the DNA environment has been compared with that of guanine. A physicochemical explanation for the mutagenic properties of oxanosine is suggested.     

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ?cysND, ?cysIJ and ?cysND-cat?cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃ ^2?) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ?cysND in the above media, but not in the ?cysIJ and ?cysND-cat?cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding l-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.     

A Theoretical Study of the cis-syn Pyrimidine Dimers in the Gas Phase and Water Cluster and a Tautomer-Bypass Mechanism for the Origin of UV-induced Mutations

Abstract

A quantum mechanical study of all cis-syn cyclobutane pyrimidine photodimers including the normal and rare tautomeric forms of bases has been performed using the ab initio method at HF/6–31G(d,p), MP2(fc)//HF/6–31G(d,p) and MP2(fc)/6–31G(d,p) levels. A puckering angle of the cyclobutyl ring and twist angle of pyrimidine rings with respect to each other is well described by these calculations. It is predicted that in the gas phase all photodimers containing the rare imino form of cytosine are more stable than those containing its normal form. The Monte Carlo simulations show that the dimer containing the imino form of cytosine is more stabilized by water cluster than that containing its amino forms. The possible biological significance stems from the fact that the cytosine in the dimer directs the incorporation of adenine in the complementary strand during replicative bypass. Data obtained point to the cytosine tautomerism as a possible mechanism for the origin of UV-induced mutation.     

Study of the Tautomerism by Experimentally and Theoretically Estimated 13C and 15N Chemical Shifts

2-Substituted 5-Me-7OH-1,2,4-Triazolo[1,5-a]pyrimidines can exist as different tautomeric forms. The tautomeric equilibrium of these compounds were determined by various NMR spectroscopic methods and quantum-chemical calculations. The tautomeric equilibria of 2-substituted 1,2,4-Triazolopyrimidines depend on the used solvent. In order to take solvent effects into account, ab initio calculations using the SCI-PCM method were carried out. The solvation energy depends on the polarity of the substituents in position 2. The comparison of chemical shifts determined by experimental methods and theoretical ab initio methods were used to definitely find out the the tautomeric equilibria of these compounds.     

Use ofN,N′-polymethylenebis(iodoacetamide) derivatives as probes for the detection of conformational differences in tubulin isotypes

Mammalian brain tubulin is anαΒ heterodimer; bothα andΒ exist in 6–7 isotypic forms which differ in their amino acid sequences. By the use of isotype-specific monoclonal antibodies, we have previously shown that we can purify theαΒ _II,αΒ _III, andaΒ _IV tubulin dimers from bovine brain. We have also observed that these isotypes differ in their distributionin vivo and their polymerization and drug-binding propertiesin vitro. We have now explored the question of whether the isotypically purified dimers differ in their overall conformation using as probes compounds of theN,N′-polymethylenebis (iodoacetamide) series which are known to form discrete intrachain cross-links inΒ-tubulin. These compounds have the structure ICH₂CONH(CH₂) _n NHCOCH₂I. One of these cross-links, designatedΒ ^s, is between cys¹² and either cys²⁰¹ or cys²¹¹. The other, designatedΒ ^*, is between cys²³⁹ and cys³⁵⁴. TheΒ ^* cross-link forms inαΒ _II andαΒ _IV but not inαΒ _III; this is not surprising in view of the fact thatαΒ _III has serine at position 239 instead of cysteine. However,αΒ _III is also unable to form theΒ ^s cross-link, although it appears to have all three cysteines which may be involved in the cross-link. This suggests that at least one of the sulfhydryls involved in the cross-link may be inaccessible inαΒ _III. Although bothαΒ _II andαΒ _IV can form theΒ ^s cross-link, the dependence on cross-linker chain length is different.αΒ _II formsΒ ^s with derivatives in whichn=2, 4, 5, 6, and 7 but not with those in whichn=3 or 10. In contrast,αΒ _IV formsΒ ^s with derivatives in whichn=2, 3, 4, 5, 6, 7, and 10. These results imply that theΒ ^s sulfhydryls are slightly more accessible inαΒ _IV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that theαΒ _II,αΒ _III, andαΒ _IV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.     

Investigation of the mechanisms of photo-induced formation of cyclobutane dimers of cytosine and 2,4-diaminopyrimidine

The mechanisms of the formation of cyclobutane dimers (CBD) of cytosine and 2,4-diaminopyrimidine were studied at the CC2 theoretical level and cc-pVDZ basis functions. Four orientations of the two monomers are explored: cys-syn, cis-anti, trans-syn, and trans-anti. The research revealed that in all cases the cyclobutane structures are formed along the ¹ππ* excited-state reaction paths of the stacked aggregates. We localized the S₁/S₀ conical intersections mediating those transformations. The results obtained agree well with the previously reported investigations on the cis-syn cyclodimer formations of other pyrimidines.     

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys₁₄₈ is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-β-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys₁₄₈ is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose:proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.     

On tautomerism of the cytosine molecule

Tautomerism of the cytosine molecule is discussed in connection with recent experimental matrix-isolation infrared spectroscopic measurements and recent ab initio calculations of relative stabilities of tautomers and of IR spectra for different tautomeric forms of the compound. Experimental IR spectra in the N-H and O-H stretching regions and in the C = O stretching region are presented for cytosine and for its several derivatives considered as model compounds. This experimental evidence, as well as the quantum-mechanical calculations (including both electron correlation and zero-point vibrational contributions), clearly indicate that two tautomers of cytosine, i.e. the amino-hydroxy and amino-oxo forms with the hydrogen atom at the N(1) position, exist in equilibrium when the cytosine molecule is isolated in an inert environment. The effect of the environment on the relative stabilities of several tautomers is also discussed briefly.     

Consequence of one-electron oxidation and one-electron reduction for aniline

Quantum-chemical calculations were performed for all possible isomers of neutral aniline and its redox forms, and intramolecular proton-transfer (prototropy) accompanied by π-electron delocalization was analyzed. One-electron oxidation (PhNH₂ – e → [PhNH₂]^+•) has no important effect on tautomeric preferences. The enamine tautomer is preferred for oxidized aniline similarly as for the neutral molecule. Dramatical changes take place when proceeding from neutral to reduced aniline. One-electron reduction (PhNH₂ + e → [PhNH₂]^-•) favors the imine tautomer. Independently on the state of oxidation, π- and n-electrons are more delocalized for the enamine than imine tautomers. The change of the tautomeric preferences for reduced aniline may partially explain the origin of the CH tautomers for reduced nucleobases (cytosine, adenine, and guanine).     

The effects of tautomerization and protonation on the adenine–cytosine mismatches: a density functional theory study

In the present work, we demonstrate the results of a theoretical study concerned with the question how tautomerization and protonation of adenine affect the various properties of adenine–cytosine mismatches. The calculations, in gas phase and in water, are performed at B3LYP/6-311++G(d,p) level. In gas phase, it is observed that any tautomeric form of investigated mismatches is more stabilized when adenine is protonated. As for the neutral mismatches, the mismatches containing amino form of cytosine and imino form of protonated adenine are more stable. The role of aromaticity on the stability of tautomeric forms of mismatches is investigated by NICS(1)_ZZ index. The stability of mispairs decreases by going from gas phase to water. It can be explained using dipole moment parameter. The influence of hydrogen bonds on the stability of mismatches is examined by atoms in molecules and natural bond orbital analyses. In addition to geometrical parameters and binding energies, the study of the topological properties of electron charge density aids in better understanding of these mispairs.     

Plant pathotoxins from Alternaria citri: Stereochemistry of the major and minor toxins

The absolute configuration of the previously characterized host-specific pathotoxins from Alternaria citri were elucidated by NMR, circular dichroism and X-ray crystallography. Each of the major tautomeric forms of the ACRL toxins are stereochemically analogous.     

Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a newin vivo amino acid biosynthesis pathway and improved wool growth

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesis cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes ofSalmonella typhimurium, cysE,cysM andcysK, and linkedcysEM,cysME andcysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and-cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and-cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cellsin vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.     

Unexpected Shift to a 4-Imino Tautomer Upon N4-Acylation of 5-Methylcytosin-1-yl Nucleoside Analogues

Abstract

In contrast with the behaviour of 5-unsubstituted cytosine nucleoside analogues, 5-methylcytosine derivatives show upon N⁴-benzoylation, commonly used as base protection in oligonucleotide synthesis, a tautomeric change of the base moiety from a 4-amino- into a 4-imino isomer. In the latter form, which is easily diagnosticized by ¹³C NMR and confirmed by X-ray data, the compounds seem to be hydrolytically less stable.     

Developmental regulation of choline sulfatase and aryl sulfatase in Neurospora crassa

Regulation of the synthesis of several enzymes of sulfur metabolism in Neurospora is a function of both metabolic regulation and the genetic control exerted by the cys-3 and scon regulatory genes. Additional control mechanisms appear to regulate the synthesis of choline sulfatase and aryl sulfatase in different developmental stages of the life cycle. The metabolic regulation of enzyme synthesis in conidia differs from that which occurs in the mycelial stage. During conidial germination and mycelial outgrowth, the synthesis of these enzymes is not coordinate but begins at different times and occurs at different rates. A rapid and early synthesis of choline sulfatase was observed during conidial germination under derepressing conditions; furthermore, synthesis of the enzyme also occurred for a brief period in germinating conidia even in the presence of repressing levels of sulfate. The results of this study suggest that several enzymes of sulfur metabolism are independently controlled by a developmental system which is superimposed upon the cys-3 regulatory mechanism. It was also found that choline sulfatase undergoes rapid turnover while aryl sulfatase is a stable species.     

The structure of hikizimycin. I. Identification of 3-amino-3-deoxy-D-glucose and cytosine as structural components

The molecular formula for hikizimycin, a new antibiotic isolated from Streptomyces A-5, is now established as C₂₁H₃₇N₅O₁₄. Evidence in support of the occurrence of 3-amino-3-deoxy-D-glucose and cytosine residues in the antibiotic molecules is presented. Methanolysis of N,N′-diacetylhikizimycin gave mainly two components. Fragment A was characterized as methyl 3-amino-3-deoxy-D-glucopyranoside by its conversion into the known methyl 3-acetamido-2,4,6-tri-O-acetyl-3-deoxy-α-D-glucopyranoside. Fragment B, on catalytic hydrogenation followed by acid hydrolysis, gave tetrahydropyrimidin-2-one, characterized as its picrate, thus establishing the presence of a cytosine residue in hikizimycin.     

Charges of phosphate groups. A role in stabilization of 2'-deoxyribonucleotides. A DFT investigation

We have analyzed the relative stabilities and Gibbs tautomeric free energy for tautomeric transitions of neutral 2'-deoxyribonucleotides and its mono- and di-protonated forms. Geometry optimizations of these nucleic acid constituents have been performed at the DFT/B3LYP level using the standard 6-31G(d) basis set. The prediction of relative stabilities, Gibbs tautomeric free energy has been made at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory. For each nucleoside four major conformers, i.e., north/anti, north/syn, south/anti, and south/syn have been taken into consideration. We have found the substantial effect of the uncompensated charge on the relative stability of 2'-deoxyribonucleotides. In particular, when the charge of 2'-deoxyribonucleotide anions is completely compensated by protons, the syn conformations have been found to be the global minima due to stabilization provided by intramolecular hydrogen bonds. However, the negative charge that appears due to the successive removal of the protons from the phosphate group destabilizes these syn conformations and stabilizes preferably the south/anti conformations (except of 2'-deoxyguanosine phosphate). Only 2'-deoxyribonucleotides, possessing south/anti and north/anti orientations, containing guanine and cytosine can contribute significantly to the rate of spontaneous point mutations due to the formation of biologically relevant amounts of 'rare' tautomers. However, we found strong influence of uncompensated negative charge for 2'-deoxyribonucleotides which possess syn conformations. Finally we have found that the proton transfer could result in the spontaneous change of 2'-deoxyribonucleotides conformations. We conclude that this phenomenon could be considered as a new way for the stabilization of 'rare' isomers for such DNA bases as cytosine and thymine.     

Reassignment of the methine resonances of D-fructose based on the carbon-13 n.m.r. spectrum of 3-O-methyl-D-fructose

Several disagreements in the ¹³C n.m.r. assignments of the methine carbons of D-fructose exist in the literature. In order to settle these inconsistencies, we examined the ¹³C n.m.r. spectrum of 3-O-methyl-D-fructose. By following the methyl induced shift in this spectrum, as compared to the parent sugar, we identified the alkylated C-3 resonance of all four tautomeric forms of D-fructose. This information, together with our previous identification of the C-5 resonances of the α- and β-forms of D-fructofuranose 6-phosphate, allow the unambiguous identification of all methine carbons of D-fructose in its ¹³C n.m.r. spectrum. The tautomeric composition of 3-O-methyl-D-fructose at 16.5°, in aqueous solution, was found to be as follows: α-pyranose 18%, β-pyranose 37%, α-furanose 11% and β-furanose 34%.