Expression of the macrolide-lincosamide-streptogramin-B-resistance methylase gene, ermE, from Streptomyces erythraeus in Escherichia coli results in N6-monomethylation and N6,N6-dimethylation of ribosomal RNA

The ermE gene was cloned from Streptomyces erythraeus into Escherichia coli on a series of plasmids. When transcribed from the lac promoter, ermE conferred high-level resistance to erythromycin and other macrolide-lincosamide-streptogramin-B (MLS) antibiotics. A methylase activity capable of N6-mono- and N6,N6-dimethylation of adenine residues in E. coli rRNA was detected in extracts of MLS-resistant cells. In addition, rRNA extracted from MLS-resistant E. coli contained N6-mono- and N6,N6-dimethylated adenine residues.     

Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: beta-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N'-triacetylchitobiose and 6'-O,N,N'-triacetylchitobiose

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (5a) was used as the glycosyl donor in a beta-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6'-O,N,N'-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N'-triacetylchitobiose (4).     

Stacking interactions of nucleobases: NMR-investigations. I. Self association of N6, N9-dimethyladenine and N6-dimethyl-N9-ethyladenine.

The selfassociation of N6,N9-dimethyladenine and N6-dimethyl-N9-ethyladenine has been studied by means of NMR technique. The thermodynamic quantities have been calculated using an isodesmic NMR model with three NMR parameters (the monomer shift deltaM and two complex shifts delta2 and delta3). The dependence of the thermodynamic quantities on the NMR parameters is discussed. Special attention is given to the determination of deltaM and its temperature dependence. Calculations with delta3 = 2 - delta2 and deltaM taken independently of temperature result in an average entropy deltaS = - 17.9 +/- 1.8 e.u. for N6,N9-dimethyladenine and deltaS = - 16.7 +/- 1.7 e.u. for N6-dimethyl-N9-ethyladenine and in an average enthalpy deltaH = - 7.2 +/- 0.6 kcal - mol-1 for both substances investigated.     

Synthesis of tritium-labelled isopenicillin N, penicillin N and 6-aminopenicillanic acid.

1. Phenoxymethylpenicillin sulphoxide 4-methoxybenzyl ester was labelled with 3H in its 2-beta-methyl group. Its specific radioactivity was 362 mCi/mmol. 2. Removal of the side chain of this compound yielded the corresponding ester of 6-aminopenicillanic acid sulphoxide and coupling of the latter with the appropriate protected alpha-aminoadipic acid gave 4-methoxybenzyloxycarbonylisopenicillin N sulphoxide di-4-methoxybenzyl ester or the corresponding derivative of penicillin N. 3. Removal of the protective groups by hydrogenolysis and reduction of the sulphoxide group yielded 3H-labelled isopenicillin N or penicillin N. 4. 3H-labelled phenoxymethylpenicillin sulphoxide was obtained by hydrogenolysis from its 4-methoxybenzyl ester. Reduction of its sulphoxide group and subsequent removal of the side chain gave 3H-labelled 6-aminopenicillanic acid.     

N6-Trimethyl-lysine metabolism. 3-Hydroxy-N6-trimethyl-lysine and carnitine biosynthesis.

Rats injected with N6-[Me-3H]trimethyl-lysine excrete in the urine five radioactively labelled metabolites. Two of these identified metabolites are carnitine and 4-trimethylammoniobutyrate. A third metabolite, identified as 5-trimethylammoniopentanoate, is not an intermediate in the biosynthesis of carnitine; the fourth and major metabolite, N2-acetyl-N6-trimethyl-lysine, is not a precursor of carnitine. The remaining metabolite (3-hydroxy-N6-trimethyl-lysine) is converted into trimethylammoniobutyrate and carnitine by rat liver slices and into trimethylammoniobutyrate by rat kidney slices. In rat liver and kidney-slice experiments, radioactivity from DL-N6-trimethyl-[1-14C]lysine and DL-N6-trimethyl-[2-14C]lysine was incorporated into N2-acetyl-N6-trimethyl-lysine and 3-hydroxy-N6-trimethyl-lysine, but not into trimethylammoniobutyrate or carnitine. A procedure was devised to purify milligram quantities of 3-hydroxy-N6-trimethyl-lysine from the urine of rats injected chronically with N6-trimethyl-lysine (100 mg/kg body wt. per day). The structure of 3-hydroxy-N6-trimethyl-lysine was confirmed chemically and by nuclear-magnetic-resonance spectrometry [Novak, Swift & Hoppel (1980) Biochem. J. 188, 521--527]. The sequence for carnitine biosynthesis in liver is: N6-trimethyl-lysine leads to 3-hydryxy-N6-trimethyl-lysine leads to leads to 4-trimethylammoniobutyrate leads to carnitine.     

Tautomerism and conformation of the promutagenic analogue N6-methoxy-2'',3'',5''-tri-O-methyladenosine

N6-Methoxy-2',3',5'-tri-O-methyladenosine crystallizes in space group P2(1)2(1)2(1) with cell dimensions a = 4.693, b = 11.412, c = 31.741 A. Least-squares refinement of diffractometer data converged at R = 0.038. The location of a hydrogen atom at N1 and the observed bond lengths and bond angles indicate unequivocally the imino tautomer of the adenine moiety. The N6-methoxy group is oriented syn to N1 and the glycosidic torsion angle XCN is -3.6 degrees, i.e. in the anti range. The furanose ring has a C2'-exo/C3'- endo pucker (P = 0.9 degrees) and is unusually flattened (tau m = 30.0 degrees). The conformations of the O-methyl groups of the ribose ring are compared with those of monomethylated nucleosides, including the biologically important 2'-O-methyl nucleosides. Evidence is presented for the existence of C-H ... N intermolecular hydrogen bonds between adenine moieties. Bearing in mind that N6-methoxyadenosine is a promutagenic analogue, the results are compared with those for the corresponding promutagenic N4-methoxycytidine. They are also discussed in relation to the tautomerism, the conformation of the N6-methoxy group, and the associated base-pairing abilities in the absence and presence of polymerases.     

Synthesis,equilibrium studies and structural characterisation of the Zn(II) complexes with trimethylene-N6,N6'-bisadenine

The new compound trimethylene-N(6),N(6')-bisadenine (L), in which two adenine molecules are linked together by a trimethylene bridge that connects the N(6) atoms, has been prepared. Reaction of L with HgCl(2) and ZnCl(2) in concentrated HCl solution leads to crystalline solids. The X-ray characterisation of the Hg(II) complex (H(2)L)[HgCl(4)].3H(2)O reveals that it is an outer-sphere complex in which the ligand is protonated at N(1) and N(1'). In contrast, the structure of the complex [H(2)L(ZnCl(3))(2)].2H(2)O shows the ligand co-ordinated to two different Zn(II) ions through the N(7) of both adenine fragments, the protons being located on the N(1) atoms. The latter compound constitutes the first crystallographic evidence of an inner sphere complex with bis-adenines and, for this reason, an equilibrium study was carried out on the Zn(II)-L-H(+) system. Potentiometric studies indicate that L is protonated in aqueous solution to form HL(+) and H(2)L(2+) with logK(H) values of 4.42 and 3.35 (25 degrees C, 0.10 M KNO(3)). The data from potentiometric titrations in the presence of Zn(2+) can be analysed considering the formation of the species LZn(2+), HLZn(3+), LZn(2)(4+) and HLZn(2)(5+), whose stability constants exceed the value expected for a monodentate interaction of the metal ion with adenine and suggest the possibility of a polydentate behaviour of L in the pH range 2.5-5.0. In contrast, spectrophotometric titrations carried out under conditions similar to those used in the synthetic work (1 M HCl) can be fitted with a model involving exclusively the H(2)LZn(4+) and H(2)LZn(2)(6+) species with logK(M) values reasonable for the interaction of Zn(II) with the N(7) of the protonated adenine fragments. Despite the H(2)LZn(2)(6+) species has a low stability, the spectrophotometric results are in agreement with its formation under the conditions in which the solid complex was prepared.     

Were there any “misassignments” among iron–sulfur clusters N4, N5 and N6b in NADH-quinone oxidoreductase (complex I)?

NADH-quinone oxidoreductase (complex I) in bovine heart mitochondria has a molecular weight of approximately 1 million Da composed of 45 distinct subunits. It is the largest energy transducing complex so far known. Bacterial complex I is simpler and smaller, but the essential redox components and the basic mechanisms of electron and proton translocation are the same. Over the past three decades, Ohnishi et al. have pursued extensive EPR studies near liquid helium temperatures and characterized most of the iron–sulfur clusters in complex I. Recently, Yakovlev et al. [G. Yakovlev, T. Reda, J. Hirst, Reevaluating the relationship between EPR spectra and enzyme structure for the iron-sulfur clusters in NADH:quinone oxidoreductase, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12720–12725] challenged Ohnishi's group by claiming that there were EPR “misassignments” among clusters N4, N5 and N6b (in order to prevent confusion, we used current consensus nomenclature, as the nickname). They claimed that we misassigned EPR signals arising from cluster N5 to cluster N4, and signals from cluster N6b to cluster N4. They also proposed that cluster N5 has (4Cys)-ligands. Based on the accumulated historical data and recent results of our site-specific mutagenesis experiments, we confirmed that cluster N5 has (1His + 3Cys)-ligands as we had predicted. We revealed that E. coli cluster N5 signals could be clearly detected at the sample temperature around 3 K with microwave power higher than 5 mW. Thus Hirst's group could not detect N5 signals under any of their EPR conditions, reported in their PNAS paper. It seems that they misassigned the signals from cluster N4 to N5. As to the claim of “misassignment” between clusters N4 and N6b, that was not a possibility because our mutagenesis systems did not contain cluster N6b. Therefore, we believe that we have not made any “misassignment” in our work.     

Synthesis of nucleoside libraries on solid support. I. N2,N6-disubstituted diaminopurine nucleosides

Starting with 2-iodo-6-chloro-9-(beta-D-ribofuranosyl)purine, a library of more than 1,300 N2,N6-polysubstituted diaminopurine nucleosides was created. The starting material was condensed with a polystyrene monomethoxytrityl resin and a pool of primary and secondary amines was used to displace the 6-chloro atom with high regioselectivity. The 2-iodo was subsequently displaced by various primary amines. Nucleosides were cleaved from the resin with hexafluoroisopropanol solutions. A majority of compounds reached a purity of more than 80% without the need for any type of purification.     

N6-substituted 9-methyladenines: a new class of adenosine receptor antagonists

A series of 15 N6-substituted 9-methyladenines have been assessed as antagonists of A2-adenosine receptor-mediated stimulation of adenylate cyclase in membranes of human platelets and rat PC12 cells and of A1-adenosine receptor-mediated inhibition of adenylate cyclases in membranes of rat fat cells and as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine to A1-adenosine receptors in rat brain membranes. N6 substitution can markedly increase the potency of 9-methyladenine at A1 receptors, while having lesser effects or even decreasing potency at A2 receptors. Effects of N6 substituents on adenosine receptor activity of the 9-methyladenines are reminiscent of effects of N6 substituents on activity of adenosine, suggesting that N6 substituted 9-methyladenines bind to adenosine receptors in the same orientation as do N6-substituted adenosines. N6-Cyclopentyl-9-methyladenine with Ki values at the A1 receptors of 1.3 microM (fat cells) and 0.5 microM (brain) is at least 100-fold more potent than 9-methyladenine (Ki 100 microM, both receptors), while at the A2 receptors KB values of 5 microM (platelets) and 25 microM (PC12 cells) make it 5-fold more potent and equipotent, respectively, compared to 9-methyladenine (KB 24 microM, both receptors). N6-Cyclopentyl and several other N6-alkyl and N6-cycloalkyl analogs are selective for A1 receptors while 9-methyladenine is the most A2 receptor selective antagonist. The N6-R- and N6-S-(1-phenyl-2-propyl)-9-methyladenines, analogous to N6-R- and N6-S-phenylisopropyladenosines, exhibit stereoselectivity at both A1 and A2 receptors. Marked differences in potency of certain N6-substituted 9-methyladenines at the A2 receptors of human platelets and rat PC12 cells provide evidence that these are not identical receptors.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Adenosine derivatives with N6-alkyl, -alkylamine or -alkyladenosine substituents as probes for the A1-receptor

Three series of N6-substituted adenosine derivatives were synthesized, having in common an unbranched alkyl chain with lengths varying from 2 to 12 methylene units, but differing in their omega-alkyl substituents: N6-n-alkyladenosines (I), N6-omega-amino-alkyladenosines (II) and alpha omega,di-(adenosin-N6-yl)alkanes (III). The compounds of the latter series combine two functional groups in one molecule. A1-receptor affinity of these compounds was measured as inhibition of [3H]PIA binding to calf brain membranes. With relatively short chain lengths, compounds in series I are the most potent. In this series, optimum activity is reached with N6-n-pentyladenosine (Ki = 0.50 nM). With short chain lengths, compounds in series II and III are 6-20-fold less potent than their congeners in series I. The potency order however is reversed with higher chain lengths. While affinity in series II and III increases strongly, reaching an optimum with the nonyl derivatives, affinity in series I decreases sharply with alkyl chains larger than 8 methylene units. Highest affinity is found with 9-amino-nonyladenosine (Ki = 0.32 nM). In general, the omega-aminoalkyl derivatives are somewhat more potent than the corresponding di-adenosinyl derivatives. Implications for the possible topography of the N6 region of the A1-receptor and the area further removed from N6 are discussed.     

Preparation and properties of N6-monobutyryl adenosine 5'-monophosphate. A major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.

The synthesis and properties of N6-monobutyryl adenosine 5'-monophosphate are described. The properties of synthesized monobutyryl nucleotide have been compared to those of a metabolite recognized in previous studies (Castagna, M. C., Palmer, W.K., and Walsh, D.A. (1977) Arch. Biochem. Biophys. 181, 46-60) as the major radioactive product produced in the liver upon perfusion with N6,O2'-dibutyryl cyclic [3H]adenosine 3':5'-monophosphate. By the criteria of cochromatography on DEAE-cellulose and in three thin layer chromatographic systems and from equivalent rates of alkaline hydrolysis, N6-monobutyryl adenosine 5'-monophosphate has been identified as a major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.     

Dialkylformamidines: depurination resistant N6-protecting group for deoxyadenosine.

Sterically hindered N6-dialkylformamidine protected deoxyadenosine is more stable to acidic depurination than N6-benzoyldeoxyadenosine and is potentially a valuable protecting group in the synthesis of deoxyoligonucleotides.     

Adenosine receptor ligands with oxygenated N6-substituents

A number of novel adenosine analogs bearing oxygenated substituents in the N6-position have been prepared and evaluated as A1 adenosine agonists. Improved conditions for the synthesis of N6-substituted adenosines and a new one pot procedure for the synthesis of 2-amino-7-oxabicyclo[2.2.1]hept-5-ene are also reported.     

Characterization of cold- and high-pressure-active polygalacturonases from a deep-sea yeast, Cryptococcus liquefaciens strain N6

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0-10 degrees C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 degrees C with a very small activation volume of -1.1 ml/mol. At 10 degrees C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme-substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme-substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.     

13C, 15N, and 31P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans

The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques. The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment. The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. In the dithionite-reduced enzyme, the ring system is bent at the N(5) position. Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group. In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design, synthesis, and biological activity of N6-substituted-4'-thioadenosines at the human A3 adenosine receptor

A large series of N6-substituted-4'-thioadenosines were synthesized starting from D-gulonic-gamma-lactone, and structure-activity relationships were studied at the human A3 and other subtypes of adenosine receptors (ARs). 2-Chloro-substituted and 2-H analogues were compared. 2-Chloro-N6-methyl-4'-thioadenosine 19b was a highly potent and selective agonist (Ki=0.8+/-0.1 nM in binding) at the A3AR, and displayed the same relative efficacy in receptor activation as a known full agonist, Cl-IB-MECA. Most of N6-substituted-4'-thioadenosines were less potent in binding than the corresponding N6-substituted-adenosines or N6-substituted-4'-thioadenosine-5'-uronamides. N6-(3-Iodobenzyl) derivative 19g was demonstrated to be an A3AR-selective partial agonist displaying a Ki value of 3.2 nM.     

Identification and characterization of N6-methyladenosine modification of circRNAs in glioblastoma

This research systematically profiled the global N6-methyladenosine modification pattern of circular RNAs (circRNAs) in glioblastoma (GBM). Based on RNA methylation sequencing (MeRIP sequencing or N6-methyladenosine sequencing) and RNA sequencing, we described the N6-methyladenosine modification status and gene expression of circRNAs in GBM and normal brain tissues. N6-methyladenosine–related circRNAs were immunoprecipitated and validated by real-time quantitative PCR. Bioinformatics analysis and related screening were carried out. Compared with those of the NC group, the circRNAs from GBM exhibited 1370 new N6-methyladenosine peaks and 1322 missing N6-methyladenosine peaks. Among the loci associated with altered N6-methyladenosine peaks, 1298 were up-regulated and 1905 were down-regulated. The N6-methyladenosine level tended to be positively correlated with circRNA expression. Bioinformatics analysis was used to predict the biological function of N6-methyladenosine–modified circRNAs and the corresponding signalling pathways. In addition, through PCR validation combined with clinical data mining, we identified five molecules of interest (BUB1, C1S, DTHD1, F13A1 and NDC80) that could be initial candidates for further study of the function and mechanism of N6-methyladenosine–mediated GBM development. In conclusion, our findings demonstrated the N6-methyladenosine modification pattern of circRNAs in human GBM, revealing the possible roles of N6-methyladenosine–mediated novel noncoding RNAs in the origin and progression of GBM.     

The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate.

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state.