2′-Deoxycoformycin: Biosynthesis and Enzymatic Conversion of 8-Keto-Deoxycoformycin to 2′-Deoxycoformycin by Streptomyces Antibioticus

Abstract

Studies on t h e biosynthesis o f the N-nucleoside antibiotics have established that the purine and pyrimidine nucleosides/nucleotides serve as the carbon and nitrogen skeleton, whereas with the C-nucleoside antibioticus, the C-N precursor forthe aglycon is either acetate or glutamate. With the pyrrolopyrimidine nucleoside antibiotics (toyocamycin, tubercidin, and sangi vamycin), either two or three carbons of the N-riboside/ribotide of GTP contribute to carbons 5 and 6 of the pyrrolering and the cyano or carboxamide group. With the naturally occurring nucleoside antibiotic containing the 1,3-diazepine seven-membered ring,2′-deoxycoformycin (dCF)(I), the precursor is not immediately obvious.     

Interactions of Potent Multisubstrate Analogue Inhibitors with Purine Nucleoside Phosphorylase from Calf Spleen—Kinetic and Spectrofluorimetric Studies

Abstract

Dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen purine nucleoside phosphorylase with potent multisubstrate analogue inhibitors were studied by kinetic and spectrofluorimetric methods.     

Simultaneous Determination of Purine and Pyrimidine Metabolites in Hprt-Deficient Cell Lines

Genetic mutations in the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), are known to cause Lesch–Nyhan syndrome and Kelley–Seegmiller syndrome. In patients, purine metabolism is different from that of normal persons. We have previously developed a method for simultaneously determining the concentration of purine and pyrimidine nucleosides and nucleotides. This system was applied to determine the concentrations of nucleosides and nucleotides in HPRT-deficient cell lines. The amount of inosine 5′-monophosphate (IMP) was different in Lesch–Nyhan syndrome, Kelley–Seegmiller syndrome, and control cell lines. The difference in the amount of IMP confirmed the mutation of the enzyme.     

Analysis of Intracellular Nucleotides by Capillary Electrophoresis–Mass Spectrometry

A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism—AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.     

Pyrimidine and Purine Analogues,Effects on Cell Cycle Regulation and the Role of Cell Cycle Inhibitors to Enhance Their Cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.     

Synthetic Studies on Formamidopyrimidines Related to Clofarabine

Degradation of clofarabine (3) in 0.9% saline solution at 100°C afforded three degradation products which were determined to be formamidopyrimidines 4–6.Compounds 4 and 5 were assigned as C_1′ anomers on the basis of one-dimensional and two-dimensional NMR experiments, whereas 6 was found to be the formamidopyrimidine lacking the sugar moiety. An improved procedure for the synthesis of formamidopyrimidines was developed, wherein benzoylated clofarabine (11) was treated with allyl chloroformate, followed by deprotection of the alloc group with catalytic Pd(PPh₃)₄ and dimedone. A synthesis of compound 6 from 4 is also described.     

Left–right asymmetries and shape analysis on Ceroglossus chilensis (Coleoptera: Carabidae)

Bilateral symmetry is widespread in animal kingdom, however most animal can deviate from expected symmetry and manifest some kind of asymmetries. Fluctuating asymmetry is considered as a tool for valuating developmental instability, whereas directional asymmetry is inherited and could be used for evaluating evolutionary development. We use the method of geometric morphometrics to analyze left/right asymmetries in the whole body, in two sites and totally six populations of Ceroglossus chilensis with the aim to infer and explain morphological disparities between populations and sexes in this species. In all individuals analyzed we found both fluctuating asymmetry and directional asymmetry for size and shape variation components, and a high sexual dimorphism. Moreover a high morphological variability between the two sites emerged as well. Differences in diet could influence the expression of morphological variation and simultaneously affect body sides, and therefore contribute to the symmetric component of variation. Moreover differences emerged between two sites could be a consequence of isolation and fragmentation, rather than a response to local environmental differences between sampling sites.     

Deriving how far structural information is transmitted through parallel homodimeric coiled‐coils: A correlation analysis of helical staggers

How local conformation is affected by local sequence is fairly well understood for alpha‐helical coiled‐coils, but less is known about how local conformation is influenced by distant features. Here, I describe an approach to detect such an effect, based on computing correlation coefficients of local out‐of‐register alignments, or so‐called “staggers” between the helices, as a function of the axial distance between the staggers. This approach requires parallel homodimers, in which each stagger can occur with two “signs,” where either one helix or the other is shifted towards the N terminus. The signs of such staggers separated by up to 12 residues are strongly correlated, indicating that the conformations of the ends of coiled‐coils are commonly influenced by attached structures. Thus, the structures of coiled‐coil residues aberrantly attached to alternative proteins, such as those resulting from leukemogenic chromosomal rearrangements, may be distinguishable from those in normal tissues, and in turn serve as targets of selective drug design. The signs of helical staggers separated by between 13 and 30 residues are moderately yet significantly correlated, indicating that some of the coiled‐coils transmit this conformational feature axially for at least 45 Å. A positive, albeit noisy, correlation also exists among tropomyosin coiled‐coils for signed staggers separated by the 40‐residue actin repeat distance, consistent with the semi‐flexible tropomyosin filament binding F‐actin and regulating skeletal muscle contraction in a partially cooperative manner. Communication of the signs of axial staggers is explained in part by minimization of main‐chain hydrogen bond deformations. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.