NMR analyses on N-hydroxymethylated nucleobases - implications for formaldehyde toxicity and nucleic acid demethylases.

Formaldehyde is produced in cells by enzyme-catalysed demethylation reactions, including those occurring on N-methylated nucleic acids. Formaldehyde reacts with nucleobases to form N-hydroxymethylated adducts that may contribute to its toxicity/carcinogenicity when added exogenously, but the chemistry of these reactions has been incompletely defined. We report NMR studies on the reactions of formaldehyde with canonical/modified nucleobases. The results reveal that hydroxymethyl hemiaminals on endocyclic nitrogens, as observed with thymidine and uridine monophosphates, are faster to form than equivalent hemiaminals on exocyclic nitrogens; however, the exocyclic adducts, as formed with adenine, guanine and cytosine, are more stable in solution. Nucleic acid demethylase (FTO)-catalysed hydroxylation of (6-methyl)adenosine results in (6-hydroxymethyl)adenosine as the major observed product; by contrast no evidence for a stable 3-hydroxymethyl adduct was accrued with FTO-catalysed oxidation of (3-methyl)thymidine. Collectively, our results imply N-hydroxymethyled adducts of nucleic acid bases, formed either by reactions with formaldehyde or via demethylase catalysis, have substantially different stabilities, with some being sufficiently stable to have functional roles in disease or the regulation of nucleic acid/nucleobase activity.

Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

Analysis of the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate by active-site mutants of Aspergillus nidulans isopenicillin N synthase.

BACKGROUND: Penicillins and cephalosporins constitute a major class of clinically useful antibiotics. A key step in their biosynthesis involves the oxidative cyclisation of delta-(Lalpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N by isopenicillin N synthase (IPNS). This chemically remarkable transformation has been extensively studied using substrate analogues. The conversion of an analogue in which the valine is replaced by alpha-aminobutyrate results in three products, two epimeric penams and a cepham. The ratio of these products in reactions catalysed by four different IPNS isozymes has been used previously to probe the thermicity of the chemical mechanism. But how IPNS restricts the products from the natural substrate to a single penam (isopenicillin N) has remained unknown. RESULTS: A key active-site residue, Leu223, identified according to a model of enzyme-substrate binding, has been altered to sterically less demanding residues. As the steric constraints on the upper part of the active site are reduced, the ratio of the beta-methyl penam to the cepham increases when the alpha-aminobutyrate-containing substrate analogue is used. These results suggest a mechanism for processing of the natural substrate in which IPNS uses steric control to restrict the conformational freedom of an intermediate such that the only product is the penam. CONCLUSIONS: Using steric pressure to control conformation, and hence to disfavour reactions leading to alternate products, is probably the result of evolutionary selection for a biologically active product at the expense of biologically inactive byproducts. It is likely that this sort of enzymatic catalysis is used in situations where substrate conversion is highly exothermic and a variety of products are possible.

Studies toward the total synthesis of the cytotoxic sponge alkaloid pyrinodemin A.

[structure: see text]. The syntheses of the proposed structure of pyrinodemin A (1) and its cis double bond positional isomer (C15'-C16') in racemic form are described. The key reaction involved an intramolecular nitrone/double bond cycloaddition. Our results suggest that neither 1 nor its double positional isomer is the correct structure of pyrinodemin A

Evidence for oxidation at C-3 of the flavonoid C-ring during anthocyanin biosynthesis.

Evidence is presented for initial oxidation at the C-3 position of the flavonoid C-ring and for two bifurcating steps during catalysis by anthocyanidin synthase.

Inhibition of human leukocyte and porcine pancreatic elastase by homologues of bovine pancreatic trypsin inhibitor.

The interactions of three BPTI homologues with human leukocyte elastase and porcine pancreatic elastase have been investigated. The principal mutation in determining the specificity of inhibition was the Lys15-Val mutation at the P1 position. An additional mutation at P3, i.e., BPTI (Lys15-Val, Pro13-Ile), increased the inhibition of HLE to a Ki = 2.5 x 10(-10) M, but decreased the inhibition of PPE, showing this to be a useful site for improving selectivity. Kinetic evidence suggests that the inhibition of HLE by BPTI homologues probably takes place by a two-step mechanism in which an isomerization step occurs after initial binding. 1H NMR spectroscopy of the BPTI (Lys15-Val) and BPTI (Lys15-Val, Pro13-Ile) mutants indicates that small conformational changes are associated with the mutations, but these are localized in the immediate vicinity of the mutation in the outer binding loop and in the inner loop connected to it through the Cys14-Cys38 disulfide bridge.

Structure and properties of TentaGel resin beads: implications for combinatorial library chemistry.

In view of the widespread use of TentaGel resin beads for the synthesis of combinatorial libraries, the properties of TentaGel resin have been examined using a combination of confocal laser microscopy and NMR spectroscopy. Evidence is presented that trypsin, a 23.5-kDa enzyme, can penetrate to the core of 90-microns TentaGel beads, and that the matrix of such beads permits molecular motion at a similar rate to that in solution. The beads act as a separate gel phase rather than as a porous solid. These conclusions have important implications for the bioassay of on-bead combinatorial chemical libraries.

Comments on the use of a dichromophoric circular dichroism assay for the identification of beta-turns in peptides.

Use of the dichromophoric CD assay for beta-turn formation in peptide sequences has been investigated. The assay involves the observation of Cotton effects in CD spectra, originating from the approach of N- and C-terminal aromatic chromophores in tetrapeptides. The approach of the chromophores was believed to be brought about by a beta-turn in the peptide structure. Our investigations were paralleled by NMR studies which revealed the presence of a previously unreported hydrogen bond in the beta-turn conformers, which appears to play a role in the generation of the observed Cotton effects. This suggests caution in the use of the CD technique alone as an assay for beta-turn conformers in peptides.

Regioselective biotransformation of the dinitrile compounds 2-, 3- and 4-(cyanomethyl) benzonitrile by the soil bacterium Rhodococcus rhodochrous LL100-21.

The cyanomethyl benzonitrile compounds used for this study contain two cyano groups: a -CH(2)CN side chain, plus a cyano group attached to the benzene ring. The ortho, meta and para -CH(2)CN substituted compounds were biotransformed using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacterium had previously been grown on the mono-nitrile compounds propionitrile, benzonitrile or acetonitrile, inducing the formation of nitrile hydrolyzing enzymes.Suspensions of R. rhodochrous LL100-21 that had been grown on propionitrile or benzonitrile converted the aliphatic group of 2-(cyanomethyl) benzonitrile (a) to the corresponding carboxylic acid, 2-(cyanophenyl) acetic acid (d) with excellent recovery of the product and no evidence for any other products. Conversely, when grown on acetonitrile the bacterium converted 2-(cyanomethyl) benzonitrile (a) to the amide derivatives 2-(cyanophenyl) acetamide (k) and 2-(cyanomethyl) benzamide (l) but only in low yields.Biotransformations of 3-(cyanomethyl) benzonitrile (b) and 4-(cyanomethyl) benzonitrile (c), by suspensions of bacteria that had been grown on benzonitrile or propionitrile, resulted in hydrolysis of the aromatic nitrile to produce 3- and 4-(cyanomethyl) benzoic acid (j) and (m), respectively, both with a high yield. Low concentrations of other products were also detected, for example the diacids 3- and 4-(carboxyphenyl) acetic acid (h) and (i).When the bacterium was grown on acetonitrile it could biotransform 3- and 4-(cyanomethyl) benzonitrile (b) and (c) to different products indicating less regiospecificity by the nitrile hydratase enzyme.Comparison of the initial rates of conversion of the aliphatic cyano side chain of 2-(cyanomethyl) benzonitrile (a) and other substituted benzonitriles indicated that electronic effects did not affect the initial rate of the reaction as they would require transmission through an SP(3) methylene carbon atom.