New function of vitamin B12: cobamide-dependent reduction of epoxyqueuosine to queuosine in tRNAs of Escherichia coli and Salmonella typhimurium.

Queuosine (Q), 7-[(4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)methyl)-7- deazaguanosine, and Q derivatives usually replace guanosine in the anticodon of tRNAs(GUN) of eubacteria and of cytoplasmic and mitochondrial tRNAs of lower and higher eucaryotes except yeasts. Q appears to be synthesized de novo exclusively in eubacteria, and the free-base queuine serves as a nutrient factor for eucaryotes. Recently, a Q derivative, oQ, containing a 2,3-epoxy-4,5-dihydroxycyclopentane ring, has been identified in Escherichia coli tRNA(Tyr). Here we show that oQ is formed when E. coli or Salmonella typhimurium is grown in glucose-salt medium. The formation of oQ was independent of molecular oxygen, and oQ-tRNAs were converted to Q-tRNAs by adding cobalamin to the growth medium. Under strictly anaerobic conditions, considerable amounts of Q were present in E. coli and S. typhimurium tRNAs when the bacteria were grown in the presence of cobalt ions with glycerol as the carbon source and fumarate as the electron acceptor. Under these conditions, the biosynthesis of cobalamin was induced. The results suggest that oQ is derived from ribose and that oQ is finally reduced to Q by a cobamide-dependent enzyme.     

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.     

tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-(14)C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations. The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.     

7-Methylguanine specific tRNA-methyltransferase from Escherichia coli.

A 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E. coli tRNA. The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-. The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B. subtilis and 0.8 moles m7G residues are formed per mole tRNA. It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.     

Distribution of the modified nucleoside Q and its derivatives in animal and plant transfer RNA''s.

The modified nucleoside, 7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, designated as Q, and its derivative, Q*, were found in tRNA's from various organisms, including several mammalian tissues, other animals such as starfish, lingula and hagfish, and wheat germ. Q isolated from rat liver tRNA was found to be identical with E. coli Q by mass spectrometry and thin-layer chromatography. Thus the rare modified nucleoside Q originally isolated from E. coli tRNA, is widely distributed in various organisms. Analysis of the mass spectrum of Q* suggested that it has a different side chain from Q.     

In Silico Analog Design for Terbinafine Against Trichophyton rubrum: A Preliminary Study

The diseases caused by dermatophytes are common among several other infections which cause serious threat to human health. It is evident that enzyme squalene epoxidase is responsible for prolonged dermatophyte infection and it is appealing to note that this enzyme is also responsible for fatty acid synthesis in these groups of fungi. In the present study, terbinafine drug which targets enzyme squalene epoxidase has been explored to design its various novel analogues. The present study suggests that many more prominent drug analogues could be constituted which may be crucial towards designing new drug candidates. In the present study, we have designed a series of such analogues viz. [(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)(naphthalen-1-ylmethyl)amine, N-[8-({[(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)amino}methyl)naphthalen-1-yl]-2-(sulfoamino) acetamide, {[4-(dihydroxyamino)-8-({[(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)amino}methyl)naphthalen-1-yl]sulfanyl}methanol and (R)-{[4-({[(2E,6R)-6,7-dimethyloct-2-en-4-yn-1-yl](methyl)amino}methyl)-5-[(hydroxysulfamoyl)amino]naphthalen-1-yl]amino}sulfinic acid. Moreover, further by molecular docking approach the binding between enzyme and designed analogues was further analysed. The present preliminary report suggested a considerably good docking interaction score of −338.75 kcal/mol between terbinafine and squalene epoxidase from Trichophyton rubrum. This preliminary study implies that few designed candidate ligands can be effectual towards the activity of this enzyme and can play crucial role in pathogenesis control of T. rubrum.     

Evidence that the nucleic acid base queuine is incorporated intact into tRNA by animal cells

Queuine (the base of queuosine, Q) catalytically reduced with tritium or deuterium yields a derivative in which the proton at C-8 (purine numbering system) has been exchanged and the cyclopentene ring has been reduced to a cyclopentane ring. Mouse fibroblast tRNA has been labeled by culturing the cells in medium supplemented with [3H]- and [2H]dihydroqueuine. Such tRNA yields, upon hydrolysis, the nucleoside dihydroqueuosine and a saccharide derivative of dihydroqueuosine. Each product has been identified unambiguously by mass spectrometry and chromatography. Both the 3H- and 2H-labeled material coeluted, and no unlabeled Q nucleoside was found. Therefore, dihydroqueuine is incorporated intact into tRNA in mammalian cells. Furthermore, fractionation of the labeled tRNA on concanavalin A-agarose, which specifically binds the mannosyl-Q-containing tRNAAsp, has shown that the dihydroqueuosine-containing tRNAAsp is mannosylated. This is the first direct evidence that queuine is incorporated intact into mammalian tRNA in vivo.     

Inhibition of leucyl-tRNA synthetase in Escherichia coli by the cytostatic 5,8-dioxo-6-amino-7-chloroquinoline.

At concentrations of 1-1.6 mug/ml, 5,8-dioxo-6-amino-7-chloroquinoline causes auxotrophy for leucine in Escherichia coli MRE 600. With increasing concentrations of this quinone additional amino acids are required for growth. The amount of leucine in the pool of free amino acids is not decreased after treatment of E. coli with the quinone. Transfer RNALeu, however, is charged with leucine less than 10% in quinone-treated cells of E. coli, whereas in control cells the degree of aminoacylation is about 85%. From these data we conclude that the quinone causes auxotrophy for leucine by interacting with the charging process of tRNALeu. Quinone was found to inhibit leucyl-tRNA synthetase activity in purified extracts of E. coli with E. coli tRNA as substrate.     

Synthesis properties of the naturally occurring N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt-6A) and other related synthetic analogs.

The naturally occurring modified nucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt6A), and the corresponding glycine analog mg6A were synthesized from N6-methyl-2',3',5'-tri-O-acetyladenosine and the appropriately blocked isocyanates derived from threonine and glycine. The natural mt6A isolated from Escherichia coli tRNA (F. Kimura-Harada et al. (1972), Biochemistry 11, 3910), from wheat embryo tRNA (R. Cunningham and M. W. Gray (1974), Biochemistry 13, 543), and from rat liver tRNA (Rogg et al. (1975), Eur. J. Biochem. 53, 115) was found to be identical with the synthetic mt6A in paper and thin-layer chromatography and electrophoresis. Several analogs of the parent 6-ureidopurine ribonucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]-L-thronine (t6A), were also prepared. Starting from 2',3',5'-tri-O-acetylguanosine and 2',3',5'-tri-O-acetylcytidine and the above isocyanates, the t6A analogs, N-[(9-beta-D-ribofuranosyl-6-oxo-1H-purin-2-yl)carbamoyl]-L-threonine (t2G) and N-[(1-beta-D-ribofuranosyl-2-oxypyrimidin-4-yl)carbamoyl]-L-threonine (t4C), were prepared. Also synthesized were the corresponding glycine analogs, g2G and g4C, from guanosine and cytidine, respectively. The 2'-deoxyribosyl analog, N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)carbamoyl]-L-threonine (2'-deoxy-t6A), and the arabinosyl derivative, N-[(9-beta-D-arabinofuranosylpurin-6-yl)carbamoyl]-L-threonine (t6AraA), were synthesized from the appropriate urethane and the requisite amino acid. The ureido group in mt6A could not be hydrolyzed by the enzymes urease, peptidase, and protease. Various chemical and biological properties of the naturally occurring mt6A and the related analogs are discussed.     

A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation

A post-translational modification affecting the translation termination rate was identified in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule. It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N:(5)-methylated. Overexpression of RF2 yields protein lacking the methylation. RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine. Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2. Thr246 decreases RF2-dependent termination efficiency compared with Ala246, especially for short peptidyl-tRNAs. Methylation of Gln252 increases the termination efficiency of RF2, irrespective of the identity of the amino acid in position 246. We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.     

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.     

Structure determination of a nucleoside Q precursor isolated from E. coli tRNA: 7-(aminomethyl)-7-deazaguanosine.

A precursor of modified nucleoside Q isolated from E. coli methyl-deficient tRNA was determined to be 7-(aminomethyl)-7-deazaguanosine. The structure was deduced by means of its chromatographic and electrophoretic mobilities, and UV and mass spectra, in addition to comparison with the synthesized authentic compound. The same molecule is also found in tRNA of an E. coli mutant selected for deficient synthesis of modified nucleosides.     

Inhibition by prostacyclin and carbacyclins of endothelin-induced DNA synthesis in cultured vascular smooth muscle cells

Effects of prostacyclin and carbacyclins on endothelin-induced DNA synthesis were investigated in vascular smooth muscle cells. DNA synthesis was estimated by [3H]thymidine incorporation. Five carbacyclins used in this report were 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo [3.3.0]oct-2-en-3-yl) pentanoic acid (TEI-7165), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo[3.3.0]oct-2-en-3- yl]pentanoate (TEI-9090), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)penta noic acid (TEI-9063), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1- nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)pentanoate (TEI-1324), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-4-hydroxy-4-methyl-1- octenyl]bicyclo[3.3.0]oct-2-en-3-yl] pentanoic acid (TEI-3356). Prostacyclin and the carbacyclins inhibited the endothelin-induced DNA synthesis within the nanomolar range. These results suggest that prostacyclin and carbacyclins are possibly effective in inhibiting the proliferation of vascular smooth muscle cells under some situations in vivo.     

The phenylalanine tRNA from Mycoplasma sp. (Kid): a tRNA lacking hypermodified nucleosides functional in protein synthesis

Phenylalanine tRNA from Mycoplasma sp. (Kid) was purified and characterized. The tRNA can be aminoacylated by phenylalanyl-tRNA synthetase from both Mycoplasma and E. coli. In a tRNA-dependent cell-free E. coli amino acid incorporating system programmed with poly U pure Mycoplasma tRNA(Phe) was fully active in promoting phenylalanine incorporation, even in direct competition with homologous E. coli tRNA(Phe). Since the Mycoplasma tRNA lacks isopentenyladenosine, or any related hypermodified nucleoside, it appears that the presence of such nucleosides in tRNA is not an absolute requirement for protein synthesis.     

Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus

Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.     

Crystal structure of archaeosine tRNA-guanine transglycosylase

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.     

Polyazacyclophanes incorporating two pyridine units and a heteroaromatic pendant group as potential cleaving agents of mRNA 5'-cap structure

Four hexaazacyclophanes, 16a-d, incorporating two pyridine units and a (pyridin-2-yl)methyl or (quinolin-2-yl)methyl pendant group at one of the ring N-atoms have been prepared. The key step of the synthesis is an intermolecular cyclization of N,N-bis{[6-(tosyloxymethyl)pyridin-2-yl]methyl}-2-nitrobenzenesulfonamide (7) with either tert-butyl bis{2-[(2-nitrophenylsulfonyl)amino]ethyl}carbamate (2a) or tert-butyl bis{3-[(2-nitrophenylsulfonyl)amino]propyl}carbamate (2b) in the presence of anhydrous Cs(2)CO(3). Removal of the acid-labile tert-butoxycarbonyl protection then allows attachment of the pendant group by reductive alkylation to the exposed secondary amino group, and deprotection of the remaining aliphatic ring N-atoms completes the synthesis. The ability of the cyclophanes and their dinuclear Cu(2+) and Zn(2+) complexes to cleave the mRNA cap structure, m(7)G(5')pppG(5') (1), has been studied.