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.

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

HP0593 DNA-(N-6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3' and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.