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.

Disordered insulin secretion in the development of insulin resistance and Type 2 diabetes.

For many years, the development of insulin resistance has been seen as the core defect responsible for the development of Type 2 diabetes. However, despite extensive research, the initial factors responsible for insulin resistance development have not been elucidated. If insulin resistance can be overcome by enhanced insulin secretion, then hyperglycaemia will never develop. Therefore, a ß-cell defect is clearly required for the development of diabetes. There is a wealth of evidence to suggest that disorders in insulin secretion can lead to the development of decreased insulin sensitivity. In this review, we describe the potential initiating defects in Type 2 diabetes, normal pulsatile insulin secretion and the effects that disordered secretion may have on both ß-cell function and hepatic insulin sensitivity. We go on to examine evidence from physiological and epidemiological studies describing ß-cell dysfunction in the development of insulin resistance. Finally, we describe how disordered insulin secretion may cause intracellular insulin resistance and the implications this concept has for diabetes therapy. In summary, disordered insulin secretion may contribute to development of insulin resistance and hence represent an initiating factor in the progression to Type 2 diabetes.

Decreasing amputation rates in patients with diabetes-a population-based study.

AIMS: To assess the changing rate of amputation in patients with diabetes over a 7-year period. METHODS: All patients undergoing lower extremity amputation in Tayside, Scotland between 1 January 2000 and 31 December 2006 were identified. Temporal linkage of cases to the diabetes database was used to ascertain which amputations were in patients with diabetes. RESULTS: The incidence of major amputations fell from 5.1 [95% confidence interval (CI) 3.8-6.4] to 2.9 (95% CI 1.9-3.8) per 1000 patients with diabetes (P < 0.05). There is a clear linear trend in the adjusted incidence of major amputation (P = 0.023 and 0.027 for age- and sex-adjusted, and duration- and sex-adjusted incidences, respectively). The adjusted incidence of total amputations followed decreased linear regression trend over the whole study period when adjusted for age and sex or diabetes duration and sex (P = 0.002). CONCLUSIONS: There has been a significant reduction in the incidence of major lower extremity amputation in patients with diabetes over the 7-year period.

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Macular oedema is not predicted by perifoveal single blot haemorrhages.

AIMS: To ascertain which perifoveal changes on digital retinal screening in diabetes predict the need for subsequent macular grid or focal laser therapy. METHODS: Between 1 January 2004 and 31 December 2005, all consecutive retinal images where any lesion was within one disc diameter of the fovea were reviewed. Patients were categorized by lesion at screening as having microaneurysm, single blot haemorrhage, multiple blot haemorrhages and exudates or circinate exudates within one disc diameter of the fovea. We compared these retinal images with the findings on slit lamp examination and the related decision for laser photocoagulation. RESULTS: Four hundred and twenty-four retinal images were identified. Of these, 52 were excluded, principally because of an interval between photography and clinic attendance of greater than 120 days, leaving 372 retinal images in the study group (313 patients). No patients with a single blot haemorrhage required immediate laser therapy at ophthalmology review compared with 13 (23%) of those with multiple blot haemorrhages and 36 (16%) of those with exudates or circinate lesions (P < 0.001). Thirty-nine patients with a single blot haemorrhage who did not require laser therapy underwent ongoing follow-up. None of these underwent laser therapy for maculopathy within the study time frame (9 months from initial screening event). CONCLUSIONS: In this study, no patients with a single blot haemorrhage within one disc diameter of the fovea on digital retinal screening required laser treatment.

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.

Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes.

Mononuclear nonheme-Fe(II)-dependent oxygenases comprise an extended family of oxidising enzymes, of which the 2-oxoglutarate-dependent oxygenases and related enzymes are the largest known subgroup. Recent crystallographic and mechanistic studies have helped to define the overall fold of the 2-oxoglutarate-dependent enzymes and have led to the identification of coordination chemistry closely related to that of other nonheme-Fe(II)-dependent oxygenases, suggesting related mechanisms for dioxygen activation that involve iron-mediated electron transfer.

Proteins of the penicillin biosynthesis pathway.

Two sequential steps are common to the biosynthesis of all penicillin-derived antibiotics: the reaction of three L-amino acids to give L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine, and the oxidation of this tripeptide to give isopenicillin N. Recent studies on the peptide synthetase and oxidase enzymes responsible for these steps have implications for the mechanisms and structures of related enzymes involved in a range of metabolic processes.

Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.

Recombinant isopenicillin N synthase from Cephalosporium acremonium was expressed in Escherichia coli and the protein was purified. After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants. Two crystal forms have been obtained with either octahedral or elongated prismatic habits. The larger octahedral crystals (0.1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a = b = 124.7 A, c = 156.9 A, and diffract X-rays to about 3.5 A resolution at synchrotrons. The crystallographic asymmetric unit contains a dimer.

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS).

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis.

Studies on the active site of deacetoxycephalosporin C synthase.

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.

Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors.

We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R. pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and west of the Andean mountains.

Betti reaction enables efficient synthesis of 8-hydroxyquinoline inhibitors of 2-oxoglutarate oxygenases.

There is interest in developing potent, selective, and cell-permeable inhibitors of human ferrous iron and 2-oxoglutarate (2OG) oxygenases for use in functional and target validation studies. The 3-component Betti reaction enables efficient one-step C-7 functionalisation of modified 8-hydroxyquinolines (8HQs) to produce cell-active inhibitors of KDM4 histone demethylases and other 2OG oxygenases; the work exemplifies how a template-based metallo-enzyme inhibitor approach can be used to give biologically active compounds.

Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans.

Recombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system. The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro- or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant. The crystals (0.5-1.0 mm overall dimensions) diffract X-rays to at least 2.0 A resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 A, b = 127.0 A, and c = 139.6 A. The asymmetric unit contains one dimer, and the solvent content of the crystals is 60%. The crystals are radiation sensitive.

delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase: the order of peptide bond formation and timing of the epimerisation reaction.

delta-L-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase catalyses the formation of the common precursor tripeptide of both the penicillin and cephalosporin antibiotics from the L-enantiomers of its constituent amino acids. Replacement of cysteine with L-O-methylserine in preparative-scale incubations led to the isolation of both L-O-methylserinyl-L-valine and L-O-methylserinyl-D-valine dipeptides. The dipeptides were characterized with the aid of authentic synthetic standards by both 1H NMR and electrospray ionization MS. A revised mechanism for ACV biosynthesis involving formation of the cysteinyl-valine peptide bond before the epimerisation of valine and subsequent condensation with the delta-carboxyl of L-alpha-aminoadipate is therefore proposed.

Delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase: isolation of L-cysteinyl-D-valine, a 'shunt' product, and implications for the order of peptide bond formation.

L-Cysteinyl-D-valine was isolated from incubations of L-glutamate, L-cysteine and L-valine with delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase and identified by 1H NMR and electrospray ionization MS. This is entirely consistent with our prior proposal (Shiau, C.-Y., Baldwin, J.E., Byford, M.F., Sobey, W.J. and Schofield, C.J. (1995) FEBS Lett. 358, 97-100) that the alpha-peptide bond between cysteine and valine is formed before the delta-peptide bond between alpha-aminoadipate and cysteine. The inclusion of L-glutamate, an analogue of L-alpha-aminoadipate, did not result in a detectable amount of tripeptide product, but did increase apparent yields of L-cysteinyl-D-valine. Conceivably, formation of the L-glutamyladenylate stimulates synthesis of the cysteinyl-valine dipeptide indirectly via a conformational change in the enzyme.

Glutamine-330 is not essential for activity in isopenicillin N synthase from Aspergillus nidulans.

The non-heme ferrous dependent oxidase isopenicillin N synthase (IPNS) catalyses the biosynthesis of isopenicillin N from a tripeptide substrate. The crystal structure of Aspergillus nidulans IPNS complexed to manganese reveals a six co-ordinate metal ligated by two water molecules and four protein ligands: His-214, His-270, Asp-216 and Gln-330 (the penultimate C-terminal residue). Modification of Gln-330 to Ala or Leu, or deletion of 2 or 6 residues from the C-terminus resulted in lowering of specific activity; no activity was observed after deletion of 8 residues. The results demonstrate that metal ligation by Gln-330 is not required for catalytic activity.

Use of electrospray mass spectrometry to directly observe an acyl enzyme intermediate in beta-lactamase catalysis.

Electrospray mass spectrometry was used to directly observe intact acyl enzyme complexes formed between a class C beta-lactamase (from Enterobacter cloacae P99) and four poor substrates/inhibitors. In each case the molecular weight difference between the unreacted and the reacted beta-lactamase was consistent with the formation of an acyl enzyme.

Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay.

Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady-state kinetic parameters for the IPNS reaction were determined.

Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry.

The use of electrospray mass spectrometry as a tool in analytical biochemistry was illustrated by determination of the molecular weights of wildtype and recombinant isopenicillin N synthase (IPNS). The molecular weight of recombinant IPNS produced using an expression system which generated soluble protein was found to be between 38,364 and 38,376 Da, ca 60 mass units higher than that of the wildtype material, consistent with the presence of an additional N-terminal glycine in the former. Observed molecular weights were all ca 70 Da higher than that calculated from sequence information, consistent with the complexion of a partially hydrated iron atom to the enzyme during analysis.