flavin-adenine-dinucleotide and purine

Triplexes with a gap in the purine strand have been shown to bind adenosine or guanosine derivatives through a combination of Watson-Crick and Hoogsteen base pairing. Rigidifying the binding site should be advantageous for affinity. Here we report that clamps delimiting the binding site have a modest effect on affinity, while bridging the gap of the purine strand can strongly increase affinity for ATP, cAMP, and FAD. The lowest dissociation constants were measured for two-strand triple helical motifs with a propylene bridge or an abasic nucleoside analog, with Kd values as low as 30 nM for cAMP in the latter case. Taken together, our data suggest that improving preorganization through covalent bridges increases the affinity for nucleotide ligands. But, a bulky bridge may also block one of two alternative binding modes for the adenine base. The results may help to design new receptors, switches, or storage motifs for purine-containing ligands.

Topics: Adenine; Adenosine Triphosphate; Amino Acid Motifs; Base Pairing; Binding Sites; Cyclic AMP; DNA; Flavin-Adenine Dinucleotide; Humans; Hydrogen Bonding; Models, Molecular; Nucleic Acid Conformation; Purines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thermodynamics

Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA.

Topics: Adenosine Triphosphate; Binding Sites; DNA; Flavin-Adenine Dinucleotide; Nucleic Acid Conformation; Oligonucleotides; Purines; Thermodynamics

Purine hydroxylase (PH) from Clostridium purinolyticum contains a labile selenium cofactor and belongs to a class of enzymes known as the selenium-dependent molybdenum hydroxylases. The presence of approximately 1.1 mol of molybdenum, 0.87 mol of selenium, and 3.3 mol of iron per mol of PH was determined by atomic absorption spectroscopy. Enzyme preparations with lower than stoichiometric amounts of selenium exhibited correspondingly lower hydroxylase activities. Bound FAD, 1 mol per mol enzyme, was confirmed by UV-vis and fluorescence spectroscopy. CMP, released by acid hydrolysis, indicated the presence of a molybdopterin cytosine dinucleotide cofactor. The fully active PH utilized NADP(+) as an electron acceptor, and kinetic analysis revealed an optimal k(cat) of 412 s(-1) using hypoxanthine as the hydroxylase substrate. Xanthine, NAD(+), and NADPH had no significant effect on this reaction rate. A selenium-independent NADPH oxidase activity was exhibited by native PH. Electron paramagnetic resonance spectroscopy revealed the presence of a Mo(V) desulfo signal, FAD radical, and 2Fe-2S centers in hypoxanthine-reduced PH. No hyperfine coupling of selenium, using (77)Se isotope-enriched PH, was observed in any of the EPR active signals studied. The appearance of the desulfo signal suggests that the ligands of Mo in selenium-dependent molybdenum hydroxylases are different from the well-studied mammalian xanthine oxidoreductases (XOR) and aldehyde oxidoreductases (AOR) and suggests a unique role for Se in catalysis.

Topics: Adenine; Clostridium; Cyanides; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Flavins; Hydrogen-Ion Concentration; Hydroxylation; Hypoxanthine; Iron; Isotopes; Kinetics; Molybdenum; NADH, NADPH Oxidoreductases; NADP; Purines; Selenium; Spectrophotometry; Xanthine Dehydrogenase

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Biochemical Phenomena; Biochemistry; Cardiomegaly; Chromatography; Coenzymes; Creatine; Creatinine; Cytosine Nucleotides; Dogs; Flavin-Adenine Dinucleotide; Guanine Nucleotides; Heart Failure; Metabolism; Myocardium; NAD; Nucleotides; Phosphates; Phosphocreatine; Pulmonary Valve Stenosis; Purines; Pyrimidine Nucleotides; Research; Uracil Nucleotides

Topics: Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Freezing; Hydrogen-Ion Concentration; Iron; Kinetics; Milk; Molybdenum; Nitrogen; Oxygen; Purines; Xanthine Oxidase; Xanthines

Topics: Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Purines; Succinate Dehydrogenase