Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine

Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential. We have employed resonance Raman spectroscopy at an excitation wavelength of 260 nm, to decipher solution structure of 9-deazaguanine (9DAG) and 9-deazahypoxanthine (9DAH). These are analogues of guanine and hypoxanthine, respectively, and have been exploited to study static complexes of nucleobase binding enzymes. Such enzymes are known to perturb pK_a of their ligands, and thus, we also determined solution structures of these analogues at two, acidic and alkaline, pH. Structure of each possible protonation state and tautomer was computed using density functional theoretical calculations. Species at various pHs were identified based on isotopic shifts in experimental wavenumbers and by comparing these shifts with corresponding computed isotopic shifts. Our results show that at physiological pH, N1 of pyrimidine ring in 9DAG and 9DAH bears a proton. At lower pH, N3 is place of protonation, and at higher pH, deprotonation occurs at N1 position. The proton at N7 of purine ring remains intact even at pH 12.5. We have further compared these results with naturally occurring nucleotides. Our results identify key vibrational modes which can report on hydrogen bonding interactions, protonation and deprotonation in purine rings upon binding to the active site of enzymes.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Synthesis of novel Schiff bases and azol-β-lactam derivatives starting from morpholine and thiomorpholine and investigation of their antitubercular,antiurease activity,acethylcolinesterase inhibition effect and antioxidant capacity

In this study, new Schiff bases and β-lactam derivatives containing morpholine and thio morpholine nuclei were synthesized. Antimicrobial, antioxidant, antimicrobial and antioxidant properties of all synthesized compounds were investigated and highly effective products were obtained. In this context, new effective structures were introduced to the literature.     

On the Detection of Unusual Points in Regression

Some procedures, based on studentized residuals and the diagonal elements of the ‘hat’ matrix, for the detection of unusual points in regression are discussed. These procedures are quite simple but effective to uncover unusual structure of the data. Some examples are given.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

RNA editing of 10 Didymium iridis mitochondrial genes and comparison with the homologous genes in Physarum polycephalum

Regions of the Didymium iridis mitochondrial genome were identified with similarity to typical mitochondrial genes; however, these regions contained numerous stop codons. We used RT-PCR and DNA sequencing to determine whether, through RNA editing, these regions were transcribed into mRNAs that could encode functional proteins. Ten putative gene regions were examined: atp1, atp6, atp8, atp9, cox1, cox2, cytb, nad4L, nad6, and nad7. The cDNA sequences of each gene could encode a functional mitochondrial protein that was highly conserved compared with homologous genes. The type of editing events and editing sequence features were very similar to those observed in the homologous genes of Physarum polycephalum, though the actual editing locations showed a variable degree of conservation. Edited sites were compared with encoded sites in D. iridis and P. polycephalum for all 10 genes. Edited sequence for a portion of the cox1 gene was available for six myxomycetes, which, when compared, showed a high degree of conservation at the protein level. Different types of editing events showed varying degrees of site conservation with C-to-U base changes being the least conserved. Several aspects of single C insertion editing events led to the preferential creation of hydrophobic amino acid codons that may help to minimize adverse effects on the resulting protein structure.     

Fungal populations of the haulm base of wheat

Abstract In a 2-year field survey, the phylloplane mycoflora on the haulm base of wheat was studied by the dilution plate technique. No significant colonisation was registered before the end of May. Cladosporium spp., 'white' and 'red' yeasts were found to be the most abundant fungi.