Selective reactions of nucleobases under biological conditions.

Pentacyanonitrosylferrate/II/ complex reacts under biological conditions (pH= 7.5, T= 25-40 degrees C, dilute solution) selectively with nucleobases. The reaction with adenine and guanine probably leads to nitrosation. A new compound formed in the reaction with adenine is prepared; both this compound and the pentacyanonitrosylferrate/II/ inhibits the multiplication of Escherichia coli.     

DNA damage recognition and repair by the murine MutY homologue

E. coli MutY excises adenine from duplex DNA when it is mispaired with the mutagenic oxidative lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG). While E. coli MutY has been extensively studied, a detailed kinetic analysis of a mammalian MutY homologue has been inhibited by poor overexpression in bacterial hosts. This current work is the first detailed study of substrate recognition and repair of mismatched DNA by a mammalian adenine glycosylase, the murine MutY homologue (mMYH). Similar to E. coli MutY, the processing of OG:A substrates by mMYH is biphasic, indicating that product release is rate-limiting. Surprisingly, the intrinsic rates of adenine removal from both OG:A and G:A substrates by mMYH are diminished ( approximately 10-fold) compared to E. coli MutY. However, similar to E. coli MutY, the rate of adenine removal is approximately nine-fold faster with an OG:A- than a G:A-containing substrate. Interestingly, the rate of removal of 2-hydroxyadenine mispaired with OG or G in duplex DNA by mMYH was similar to the rate of adenine removal from the analogous context. In contrast, 2-hydroxyadenine removal by E. coli MutY was significantly reduced compared to adenine removal opposite both OG and G. Furthermore, dissociation constant measurements with duplexes containing noncleavable 2'-deoxyadenosine analogues indicate that mMYH is less sensitive to the structure of the base mispaired with OG or G than MutY. Though in many respects the catalytic behavior of mMYH is similar to E. coli MutY, the subtle differences may translate into differences in their in vivo functions.     

Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.     

YhdJ, a nonessential CcrM-like DNA methyltransferase of Escherichia coli and Salmonella enterica

The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the alpha-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.     

Adenine Deaminase Activity of the yicP Gene Product of Escherichia coli

During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60°C. The apparent K_m for adenine was 0.8 mM.     

Adenine deaminase activity of the yicP gene product of Escherichia coli.

During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60 degrees C. The apparent Km for adenine was 0.8 mM.     

X-ray crystallographic analysis of the structural basis for the interaction of pokeweed antiviral protein with guanine residues of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP), which enzymatically removes a single adenine base from a conserved, surface exposed loop sequence of ribosomal rRNA. We now present unprecedented experimental evidence that PAP can release not only adenine but guanine as well from Escherichia coli rRNA, albeit at a rate 20 times slower than for adenine. We also report X-ray structure analysis and supporting modeling studies for the interactions of PAP with guanine. Our modeling studies indicated that PAP can accommodate a guanine base in the active site pocket without large conformational changes. This prediction was experimentally confirmed, since a guanine base was visible in the active site pocket of the crystal structure of the PAP-guanine complex.     

Nucleotide binding drives conformational changes in the isolated alpha and beta subunits of the F(1)-ATPase from Escherichia coli

The modeling of the rotatory mechanism performed by the F(1)-ATPase complex during ATP synthesis shows that the beta, but not the alpha subunit, undergoes large conformational changes that depend on the occupancy of the catalytic site. Here we determined by fluorescence spectroscopy the changes in tertiary structure and hydrophobic exposed area of the isolated alpha and beta subunits of the F(1)-ATPase complex from Escherichia coli upon adenine nucleotide binding. The results show that in the absence of intersubunit contacts, the two subunits exhibit markedly similar conformational movements.     

A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis.

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin-biotin detection. The main complex (mol. wt. congruent to 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypothesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.     

Excision Repair of 2,5-Diaziridinyl-1,4-Benzoquinone (DZQ)-DNA Adduct by Bacterial and Mammalian 3-Methyladenine-DNA Glycosylases

The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.     

Mode of Nicotinamide Adenine Dinucleotide Utilization by Escherichia coli

Exogenous nicotinamide adenine dinucleotide is not utilized per se by Escherichia coli, but is converted to nicotinamide and thence to nicotinamide adenine dinucleotide via nicotinate.     

Identification, purification, and characterization of Escherichia coli virus T1 DNA methyltransferase

An Escherichia coli virus T1-induced DNA methyltransferase was identified by activity gel analysis in homogenates of infected E. coli DNA-adenine-methylation-deficient strains. Although the Mr of this protein (31,000) is in the same range as that of the E. coli DNA adenine methyltransferase, the two proteins are not closely related; the E. coli dam gene does not hybridize with T1 DNA. Selective conditions for measurement of the T1 activity were developed, and the enzyme was purified to functional homogeneity, as shown by activity analysis in polyacrylamide gels. Requirements for optimal activity of the viral enzyme were determined to be pH 6.9, ionic strengths below 0.1 M KCl, and a temperature between 40 and 43 degrees C. The Km for S-adenosyl-L-methionine is 4.9 microM. The purified T1 DNA methyltransferase is capable of methylating adenine in 5'-GATC-3' sites in vitro.     

Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.

The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits.     

Isolation of mutants with impaired retroinhibition of histidine biosynthesis

The Escherichia coli, strain possessing purF, deoD and add mutations converts exogenous adenine into guanine nucleotides exclusively by the pathway coupled with histidine biosynthesis. When grown on adenine, this strain demonstrated sensitivity to histidine, thus making it possible to select histidine-resistant hisGR mutants with ATP-phosphoribosyltransferase desensibilized for histidine. The hisGR mutations were obtained in two his operons introduced into the his operon-sensitive E. coli strain: his operon of Salmonella typhimurium incorporated in DNA and his operon of E. coli on the F'episome. In both cases, the hisGR mutants obtained were shown to excrete histidine.     

The DNA adenine methyltransferase (dam+) gene of bacteriophage T4 reverses the mutator phenotype of an Escherichia coli dam mutant.

The mutator phenotype of Escherichia coli dam mutants was found to be reversed by introduction of the bacteriophage T4 gene for DNA adenine methyltransferase. This precludes a direct role for the E. coli DNA adenine methyltransferase in mismatch repair, in addition to its role in strand discrimination, as suggested by earlier studies (S. L. Schlagman, S. Hattman, and M. G. Marinus, J. Bacteriol. 165:896-900, 1986).     

Sulfate-reducing pathway in Escherichia coli involving bound intermediates.

Although a sulfate-reducing pathway in Escherichia coli involving free sulfite and sulfide has been suggested, it is shown that, as in Chlorella, a pathway involving bound intermediates is also present. E. coli extracts contained a sulfotransferase that transferred the sulfonyl group from a nucleosidephosphosulfate to an acceptor to form an organic thiosulfate. This enzyme was specific for adenosine 3'-phosphate 5'-phosphosulfate, did not utilize adenine 5'-phosphosulfate, and transferred to a carrier molecule that was identical with thioredoxin in molecular weight and amino acid composition. In the absence of thioredoxin, only very low levels of the transfer of the sulfo group to thiols was observed. As in Chlorella, thiosulfonate reductase activity that reduced glutathione-S-SO3- to bound sulfide could be detected. In E. coli, this enzyme used reduced nicotinamide adenine dinucleotide phosphate and Mg2+, but did not require the addition of ferredoxin or ferredoxin nicotinamide adenine dinucleotide phosphate reductase. Although in Chlorella the thiosulfonate reductase appears to be a different enzyme from the sulfite reductase, the E. coli thiosulfonate reductase and sulfite reductase may be activities of the same enzyme.     

N-Alkoxysulfamide,N-hydroxysulfamide,and sulfamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases

A series of sulfamate surrogates of methionyl and isoleucyl adenylate have been investigated as MetRS and IleRS inhibitors by modifications of the sulfamate linker and adenine moieties. The discovery of 2-iodo Ile-NHSO(2)-AMP (58) as a potent Escherichia coli IleRS inhibitor revealed that a significant hydrophobic interaction between the 2-substituent of Ile-NHSO(2)-AMP and the adenine binding site of IleRS provided its high potency to the enzyme.     

Effect of 2,6-diaminopurine resistance mutations on adenine and adenosine assimilation by cells of an adenine-dependent strain of Escherichia coli K-12]

In purine-requiring strain of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pur, pup) mutants (designated apt) have been obtained that are resistant to 2,6-diaminopurine on guanine-containing medium and incapable to utilize adenine for their growth at 42degreesC, but they are still sensitive to the analogue and can utilize adenine at 28degreesC. It has been shown that the introduction of the corresponding apt mutations in the genome of adenine-requiring strains impaired the ability of these strains to grow on both adenine and adenosine at 42degreesC.     

Insight into the functional consequences of inherited variants of the hMYH adenine glycosylase associated with colorectal cancer: complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding mutated E.coli enzymes

The oxidized guanine lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair. The Escherichia coli adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated adenine residues from OG:A mismatches. Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of familial adenomatous polyposis (FAP), which is typically associated with mutations in the adenomatous polyposis coli (APC) gene. Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D. These positions are highly conserved in MutY across phylogeny. The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to complement the activity of E.coli MutY in mutY((-)) E.coli. In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the adenine glycosylase activity of the enzymes. In addition, evaluation of substrate affinity using a substrate analog, 2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G. Importantly, adenine removal with both the mutant and WT E.coli enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in tumors of Family N. Interestingly, the magnitude of the reduced activity of the E.coli mutant enzymes relative to the WT enzyme was magnified in the "hotspot" sequence context. If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene. The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH enzymes to recognize and repair OG:A mismatches.