The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Hepatitis C virus (HCV ) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site(IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly.

Presence of Nicotinamide Adenine Dinucleotide–Independent Haemophilus paragallinarum in Mexico

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent—growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3 wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NADindependent H. paragallinarum of serovar B has been reported. Abbreviations: NAD ¼ nicotinamide adenine dinucleotide; NAM ¼ nicotinamide; PCR ¼ polymerase chain reaction; TM ¼ complete growth medium without chicken serum or nicotinamide adenine dinucleotide; TM/SN ¼ complete growth medium that contains both chicken serum and nicotinamide adenine dinucleotide

Studies on 1-methyl adenine transfer RNA methyltransferase of Mycobacterium smegmatis

The presence of 1-methyl adenine in transfer RNA is a feature that Mycobacterium smegmatis shares with only a few other prokaryotes. The enzyme 1-methyl adenine tRNA methyl transferase from this source has been purified and the preliminary results show the presence of two activity peaks with different substrate specificity.

Molecular structure of nicotinamide adenine dinucleotide

NICOTINAMIDE adenine dinucleotide (NAD) has a fundamental role in metabolic processes as an electron transport molecule. Although its chemical structure was elucidated1 in 1934, its detailed conformation remains still to be established in spite of numerous physicochemical applications2. NAD analogues with a variety of substitutions on the bases are known to retain considerable activity of the natural coenzyme as long as the pyrophosphate diester group has been retained3,4. The geometry of this backbone moiety is therefore indispensable to our understanding of the conformation and function of the coenzyme. We have so far no experimental evidence on this in NAD or any other nucleotide coenzyme molecule. X-ray studies have been possible only on those analogues5,6 where the nicotinamide and adenine rings are linked by a trimethylene bridge. The results are conflicting and it is difficult to use them to provide a structural basis for the NAD molecule itself, particularly as the phosphate backbone is absent from these analogues.

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of reduced nicotinamide-adenine dinucleotide in the presence of formylphenylacetic acid ethyl ester

The oxidase-peroxidase from Datura innoxia which catalyses the oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid was also found to catalyse the oxidation of NADH in the presence of Mn2+ and formylphenylacetic acid ethyl ester. NADH was not oxidized in the absence of formylphenylacetic acid ethyl ester, although formylphenylacetonitrile or phenylacetaldehyde could replace it in the reaction. The reaction appeared to be complex and for every mol of NADH oxidized 3-4 g-atoms of oxygen were utilized, with a concomitant formation of approx. 0.8 mol of H2O2, the latter being identified by the starch-iodide test and decomposition by catalase. Benzoylformic acid ethyl ester was also formed in the reaction, but in a nonlinear fashion, indicating a lag phase. In the absence of Mn2+, NADH oxidation was not only very low, but itself inhibited the formation of benzoylformic acid ethyl ester from formylphenylacetic acid ethyl ester. A reaction mechanism for the oxidation of NADH in the presence of formylphenylacetic acid ethyl ester is proposed.

Characterization of an N6 adenine methyltransferase from Helicobacter pylori strain 26695 which methylates adjacent adenines on the same strand

Genomic sequences of Helicobacter pylori strains 26695, J99, HPAGI and G27 have revealed an abundance of restriction and modification genes. hp0050, which encodes an N6 adenine DNA methyltransferase, was cloned, overexpressed and purified to near homogeneity. It recognizes the sequence 5'-GRRG-3' (where R is A or G) and, most intriguingly, methylates both adenines when R is A (5'-GAAG-3'). Kinetic analysis suggests a nonprocessive (repeated-hit) mechanism of methylation in which HP0050 methyltransferase methylates one adenine at a time in the sequence 5'-GAAG-3'. This is the first report of an N6 adenine DNA methyltransferase that methylates two adjacent residues on the same strand. Interestingly, HP0050 homologs from two clinical strains of H. pylori (PG227 and 128) methylate only 5'-GAGG-3' compared with 5'-GRRG-3' in strain 26695. HP0050 methyltransferase is highly conserved as it is present in more than 90% of H. pylori strains. Inactivation of hp0050 in strain PG227 resulted in poor growth, suggesting its role in the biology of H. pylori. Collectively, these findings provide impetus for exploring the role(s) of this conserved DNA methyltransferase in the cellular processes of H. pylori.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Nucleotidases in plants *1, , *2: III. Effect of metabolites on the enzyme hydrolyzing flavine adenine dinucleotide (FAD) from Phaseolus radiatus

The highly purified enzyme from mung bean seedlings hydrolyzing FAD at pH 9.4 and temperature 49 °, functioned with an initial fast rate followed by a second slower rate. The activity was linear with enzyme concentration over a small range of concentration and was dependent on the time of incubation. Inhibition of enzyme activity with increasing concentrations of AMP was sigmoid;concentrations less than 1 × 10−6 M were without effect, concentrations between 1 × 10−6 and 8 × 10−5 M inhibited by 20% and concentrations beyond 8 × 10−5 Image caused progressive inhibition. Concentrations beyond 1 × 10−3 Image inhibited the activity completely. Preincubation of the enzyme with PCMB or NEM, or aging, or reversible denaturation with urea abolished the inhibitory effect of AMP at concentrations lower than 8 × 10−6 Image . The aged enzyme could be reactivated by ADP.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

Studies of the enzymes involved in nicotinamide adenine dinucleotide metabolism in Aspergillus niger

The enzyme nicotinamide amidase (nicotinamide amidohydrolase) was purified 57-fold from Aspergillus niger. The purified preparation was specific towards its substrate nicotinamide and did not deamidate NADP, NAD, NMN, N′-methyl nicotinamide, asparagine, glutamine, benzamide, α-naphthaleneamide and indoleacetamide. The asparagine, glutamine, benzamide, α-naphthaleneamide and indoleacetamide.vThe optimum pH was found to be 7.5. Temperature optimum was 40°. It had a Km value of 6.504 · 10−4 M towards nicotinamide. The enzyme exhibited Mg2+ ion requirement for its optimum activity. NAD-glycohydrolase (EC 3.2.2.5) was purified 109-fold from the mold. A. niger. The enzyme preparation was active only towards NAD and NADP and did not attack NMN, N′-methylnicotinamide and NADH. The Km value for NAD was found to be 7.693 · 10−6 M. The enzyme did not require any metal ion for its activity. It is suggested that A. niger will serve a better source for a large scale preparation of NAD-glycohydrolase than the Neurospora mold. The biological role of both NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD level has been discussed. It is, further, observed that NAD did not exert its feedback control on nicotinamide amidase at least in A. niger.

Crystal structure of an intermolecular 2:1 complex between adenine and thymine.Evidence for both Hoogsteen and `quasi-Watson-Crick' interactions

The titled complex, obtained by co-crystallization (EtOH/25 degrees C),is apparently the only known complex of the free bases. Its crystal structure, as determined by X-ray diffraction at both 90 K and 313 K, showed that one A-T pair involves a Hoogsteen interaction, and the other a Watson-Crick interaction but only with respect to the adenine unit. The absence of a clear-cut Watson-Crick base pair raises intriguing questions about the basis of the DNA double helix. (C) 2010 Elsevier Ltd. All rights reserved.