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.

Can patients apply the Ottawa ankle rules to themselves?

OBJECTIVE: To determine whether patients with an ankle injury obtained the same results as clinicians when applying the Ottawa ankle rules (a validated clinical decision rule) to themselves. METHODS: Patients aged >15 years presenting to an inner city emergency department within 48 h of an ankle injury were asked to assess their own injury using the Ottawa ankle rules. The results of their self-assessment were compared with those of a treating clinician. RESULTS: Poor interobserver agreement was found between patients and clinicians. CONCLUSIONS: Making the Ottawa ankle rule more widely available to the general public is unlikely to reduce healthcare demand. Indeed, given the apparently low specificity of the rule, demand could actually increase as a result.

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.

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.

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.

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.

Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A.

Phytanoyl-CoA 2-hydroxylase (PAHX), an iron(II) and 2-oxoglutarate-dependent oxygenase, catalyses an essential step in the mammalian metabolism of beta-methylated fatty acids. Phytanoyl-CoA was synthesised and used to develop in vitro assays for PAHX. The product of the reaction was confirmed as 2-hydroxyphytanoyl-CoA by NMR and mass spectrometric analyses. In accord with in vivo analyses, hydroxylation of both 3R and 3S epimers of the substrate was catalysed by PAHX. Both pro- and mature- forms of PAHX were fully active.

Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex.

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.

Oxidative degradation of bilirubin produces vasoactive compounds.

Subarachnoid haemorrhage is often followed by haemolysis and concomitant oxidative stress, and is frequently complicated by pathological vasoconstriction or cerebral vasospasm. It is known that upregulation of haem oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here we report biomimetic studies aimed at modelling pathological conditions leading to oxidative degradation of BR. Oxidative degradation products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biological activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biological activity was observed with vasoactive cerebrospinal fluid from subarachnoid haemorrhage patients. Three degradation products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivatives, 4-methyl-5-oxo-3-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegradation product of biliverdin. Possible mechanisms of oxidative degradation of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degradation products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathological vasoconstriction associated with haemorrhage.

Mechanistic insights into the inhibition of serine proteases by monocyclic lactams.

Although originally discovered as inhibitors of pencillin-binding proteins, beta-lactams have more recently found utility as serine protease inhibitors. Indeed through their ability to react irreversibly with nucleophilic serine residues they have proved extraordinarily successful as enzyme inhibitors. Consequently there has been much speculation as to the reason for the general effectiveness of beta-lactams as antibacterials or inhibitors of hydrolytic enzymes. The interaction of analogous beta- and gamma-lactams with a serine protease was investigated. Three series of gamma-lactams based upon monocyclic beta-lactam inhibitors of elastase [Firestone, R. A. et al. (1990) Tetrahedron 46, 2255-2262.] but with an extra methylene group inserted between three of the bonds in the ring were synthesized. Their interaction with porcine pancreatic elastase and their efficacy as inhibitors were evaluated through the use of kinetic, NMR, mass spectrometric, and X-ray crystallographic analyses. The first series, with the methylene group inserted between C-3 and C-4 of the beta-lactam template, were readily hydrolyzed but were inactive or very weakly active as inhibitors. The second series, with the methylene group between C-4 and the nitrogen of the beta-lactam template, were inhibitory and reacted reversibly with PPE to form acyl-enzyme complexes, which were stable with respect to hydrolysis. The third series, with the methylene group inserted between C-2 and C-3, were not hydrolyzed and were not inhibitors consistent with lack of binding to PPE. Comparison of the crystal structure of the acyl-enzyme complex formed between PPE and a second series gamma-lactam and that formed between PPE and a peptide [Wilmouth, R. C., et al. (1997) Nat. Struct. Biol. 4, 456-462.] reveals why the complexes formed with this series were resistant to hydrolysis and suggests ways in which stable acyl-enzyme complexes might be obtained from monocyclic gamma-lactam-based inhibitors.