[FeFe] hydrogenase: protonation of {2Fe3S} systems and formation of super-reduced hydride states.

The synthesis and crystallographic characterization of a complex possessing a well-defined {2Fe3S(µ-H)} core gives access to a paramagnetic bridging hydride with retention of the core geometry. Chemistry of this 35-electron species within the confines of a thin-layer FTIR spectro-electrochemistry cell provides evidence for a unprecedented super-reduced Fe(I)(µ-H)Fe(I) intermediate.

Glyconanoparticles for the plasmonic detection and discrimination between human and avian influenza virus.

A plasmonic bioassay for the specific detection of human influenza virus has been developed based on gold nanoparticles functionalised with a designed and synthesised thiolated trivalent α2,6-thio-linked sialic acid derivative. The glyconanoparticles consist of the thiolated trivalent α2,6-thio-linked sialic acid derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface. Varying ratios of the trivalent α2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of 25:75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the trivalent α2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min. Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyconanoparticles. A comparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functionalised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to discriminate between human (α2,6 binding) and avian (α2,3 binding) RG14 (H5N1) influenza virus highlighting the binding specificity of the trivalent α2,6-thio-linked sialic acid ligand.

Structure of Streptomyces maltosyltransferase GlgE, a homologue of a genetically validated anti-tuberculosis target.

GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (ß/α)(8) fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.

Paramagnetic bridging hydrides of relevance to catalytic hydrogen evolution at metallosulfur centers.

Paramagnetic hydrides are likely intermediates in hydrogen-evolving enzymic and molecular systems. Herein we report the first spectroscopic characterization of well-defined paramagnetic bridging hydrides. Time-resolved FTIR spectroelectrochemical experiments on a subsecond time scale revealed that single-electron transfer to the µ-hydride di-iron dithiolate complex 1 generates a 37-electron valence-delocalized species with no gross structural reorganization of the coordination sphere. DFT calculations support and (1)H and (2)H EPR measurements confirmed the formation an S = ½ paramagnetic complex (g = 2.0066) in which the unpaired spin density is essentially symmetrically distributed over the two iron atoms with strong hyperfine coupling to the bridging hydride (A(iso) = -75.8 MHz).

Sugar nucleotide recognition by Klebsiella pneumoniae UDP-D-galactopyranose mutase: fluorinated substrates, kinetics and equilibria.

A series of selectively fluorinated and other substituted UDP-D-galactose derivatives have been evaluated as substrates for Klebsiella pneumoniae UDP-D-galactopyranose mutase. This enzyme, which catalyses the interconversion of the pyranose and furanose forms of galactose as its UDP adduct, is a prospective drug target for a variety of microbial infections. We show that none of the 2''-, 3''- or 6''-hydroxyl groups of UDP-D-galactopyranose are essential for substrate binding and turnover. However, steric factors appear to play an important role in limiting the range of substitutions that can be accommodated at C-2'' and C-6'' of the sugar nucleotide substrate. Attempts to invert the C-2'' stereochemistry from equatorial to axial, changing D-galacto- to D-talo-configuration, in an attempt to exploit the higher percentage of furanose at equilibrium in the talo-series, met with no turnover of substrate.

Evidence for communality in the primary determinants of CYP74 catalysis and of structural similarities between CYP74 and classical mammalian P450 enzymes.

In silico structural analysis of CYP74C3, a membrane-associated P450 enzyme from the plant Medicago truncatula (barrel medic) with hydroperoxide lyase (HPL) specificity, showed that it had strong similarities to the structural folds of the classical microsomal P450 enzyme from rabbits (CYP2C5). It was not only the secondary structure predictions that supported the analysis but site directed mutagenesis of the substrate interacting residues was also consistent with it. This led us to develop a substrate-binding model of CYP74C3 which predicted three amino acid residues, N285, F287, and G288 located in the putative I-helix and distal haem pocket of CYP74C3 to be in close proximity to the preferred substrate 13-HPOTE. These residues were judged to be in equivalent positions to those identified in SRS-4 of CYP2C5. Significance of the residues and their relevance to the model were further assessed by site directed mutagenesis of the three residues followed by EPR spectroscopic and detailed kinetic investigations of the mutated proteins in the presence and absence of detergent. Although point mutation of the residues had no effect on the haem content of the mutated proteins, significant effects on the spin state equilibrium of the haem iron were noted. Kinetic effects of the mutations, which were investigated using three different substrates, were dramatic in nature. In the presence of detergent with the preferred substrate (13-HPOTE), the catalytic center activities and substrate binding affinities of the mutant proteins were reduced by a factor of 8-32 and 4-12, respectively, compared with wild-type--a two orders of magnitude reduction in catalytic efficiencies. We believe this is the first report where primary determinants of catalysis for any CYP74 enzyme, which are fully consistent with our model, have been identified. Our working model predicts that N285 is close enough to suggest that a hydrogen bond with the peroxy group of the enzyme substrate 13-HPOTE is warranted, whereas significance of F287 may arise from a strong hydrophobic interaction between the alkyl group(s) of the substrate and the phenyl ring of F287. We believe that G288 is crucial because of its size. Any other residue with a relatively bulky side chain will hinder the access of substrate to the active site. The effects of the mutations suggests that subtle protein conformational changes in the putative substrate-binding pocket regulate the formation of a fully active monomer-micelle complex with low-spin haem iron and that structural communication exists between the substrate- and micelle-binding sites of CYP74C3. Conservation in CYP74 sequence alignments suggests that N285, F287, and G288 in CYP74C3 and the equivalent residues at positions in other CYP74 enzymes are likely to be critical to catalysis. To support this we show that G324 in CYP74D4 (Arabidopsis AOS), equivalent to G288 in CYP74C3, is a primary determinant of positional specificity. We suggest that the overall structure of CYP74 enzymes is likely to be very similar to those described for classical P450 monooxygenase enzymes.

Unexpected dependence on pH of NO release from Paracoccus pantotrophus cytochrome cd1.

A previous study of nitrite reduction by Paracoccus pantotrophus cytochrome cd(1) at pH 7.0 identified early reaction intermediates. The c-heme rapidly oxidised and nitrite was reduced to NO at the d(1)-heme. A slower equilibration of electrons followed, forming a stable complex assigned as 55% cFe(III)d(1)Fe(II)-NO and 45% cFe(II)d(1)Fe(II)-NO(+). No catalytically competent NO release was observed. Here we show that at pH 6.0, a significant proportion of the enzyme undergoes turnover and releases NO. An early intermediate, which was previously overlooked, is also identified; enzyme immediately following product release is a candidate. However, even at pH 6.0 a considerable fraction of the enzyme remains bound to NO so another component is required for full product release. The kinetically stable product formed at the end of the reaction differs significantly at pH 6.0 and 7.0, as does its rate of formation; thus the reaction is critically dependent on pH.

Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein.

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO(2) and H(2). The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation.

Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer-micelle association.

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/K(m) of up to 1.6x10(8) M(-1) x s(-1) is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association.

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.

Thermally induced structural changes in glycinin, the 11S globulin of soya bean (Glycine max)--an in situ spectroscopic study.

The thermal denaturation behaviour of glycinin solutions has been studied in situ as a function of ionic strength using various spectroscopic methods. Changes in secondary structure occurred at temperatures above 60 degrees C, well before the onset of gelation. Even after heating to 95 degrees C, much of the native beta-sheet structure of glycinin was retained, as indicated by the amide I peak maximum at 1635 cm(-1) in the Fourier transformed infrared (FT-IR) spectrum. This was accompanied by an increase in the 1625 cm(-1) band, indicative of the formation of intermolecular beta-sheet associated with protein aggregation. Nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of highly mobile regions in glycinin comprising predominantly of Gln and Glu residues, corresponding to mobile regions previously identified by crystallographic studies. There was also evidence of a hydrogen-bonded structure within this mobile region, which may correspond to an alpha-helical region from Pro(256) to (or just before) Pro(269) in proglycinin. This structure disappeared at 95 degrees C, when heat-set gel formation occurred, as indicated by a sudden broadening and weakening of the NMR signal. Otherwise the NMR spectrum changed little during heating, emphasising the remarkable thermal stability of glycinin. It is proposed that during heating the core beta-barrel structure remains intact, but that the interface between the beta-domains melts, revealing hydrophobic faces which may then form new structures in a gel-network. As Cys(45), which forms the disulfide with Cys(12) linking the acidic and basic polypeptides, is found in this interface, such a rearrangement of the individual beta-domains could be accompanied by cleavage of this disulfide bond, as is observed experimentally. Such information contributes to our understanding the aggregative behaviour of proteins, and hence develops knowledge-based strategies for controlling and manipulating it.

Density and microviscosity studies of palm oil/water emulsions.

Electron paramagnetic resonance (EPR) and densitometry were used to measure the temperature- and rate-dependent formation of fat crystals in emulsion droplets in hardened palm kernel oil in water emulsions. The solid fat content in emulsions can be critical for the functionality of the emulsions in a wide variety of applications. Therefore, new and accessible methods are needed to monitor solid fat content in order to control the functional properties of these emulsions. EPR measurements showed that the microviscosity within the oil droplets could be measured as a function of temperature and that the storage temperature below the main fat melting point is crucial for an increase in the microviscosity, due to fat solidification. The microviscosity of the oil droplets could be an important parameter for defining functional performance (e.g., rheology and partial coalescence). Density measurements provided a simple and accurate method for monitoring changes in phase state of the oil. The phase behavior agreed well with the EPR results, demonstrating that accurate density measurements could prove to be a valuable tool for monitoring fat crystallization processes.

Bacillus subtilis YxaG is a novel Fe-containing quercetin 2,3-dioxygenase.

The Bacillus subtilis genome contains genes for three hypothetical proteins belonging to the bicupin family, two of which we have previously shown to be Mn(II)-dependent oxalate decarboxylases. We have now shown that the third, YxaG, exhibits quercetin 2,3-dioxygenase activity and that it contains Fe ions. This contrasts with the eukaryotic enzyme which contains a Cu ion. YxaG is the first prokaryotic carbon monoxide-forming enzyme that utilises a flavonol to be characterised and is only the second example of a prokaryotic dioxygenolytic carbon monoxide-forming enzyme known to contain a cofactor. It is proposed to rename the B. subtilis gene qdoI.

Thiamine biosynthesis in Escherichia coli: isolation and initial characterisation of the ThiGH complex.

In Escherichia coli, two of the proteins required for the biosynthesis of the thiazole moiety of thiamine (vitamin B(1)) are ThiG and ThiH, encoded as part of the thiCEFSGH operon. In this study, a C-terminally hexahistidine-tagged ThiH (ThiH-His) was expressed in E. coli as a soluble protein from thiGH-His-tag and thiFSGH-His-tag-bearing plasmids. When isolated under anaerobic conditions, ThiG and ThiH-His co-purify as a large multimeric non-covalent complex. Electron paramagnetic resonance and UV-visible spectroscopy together with iron and sulfide analyses revealed the presence of an iron-sulfur cluster within this complex.

Transient FTIR spectroelectrochemical and stopped-flow detection of a mixed valence (Fe(I)-Fe(II)) bridging carbonyl intermediate with structural elements and spectroscopic characteristics of the di-iron sub-site of all-iron hydrogenase.

Iron(I) in biology?: one-electron oxidation of an (Fe(I)-Fe(I)) carbonyl cyanide precursor bearing a proximal thioether group leads to an (Fe(I)-Fe(II)) bridging carbonyl transient with spectral features similar to the di-iron sub-site of the CO inhibited paramagnetic centre of all-iron hydrogenase.

An expedient enzymatic route to isomeric 2-, 3- and 6-monodeoxy-monofluoro-maltose derivatives.

2-Deoxy-2-fluoro-d-glucose, 3-deoxy-3-fluoro-D-glucose and 6-deoxy-6-fluoro-D-glucose were converted into the corresponding maltose derivatives using Arabidopsis thaliana DPE2-mediated trans-glycosylation reaction with glycogen acting as a glucosyl donor. (19)F NMR spectroscopy proved to be a valuable tool for monitoring the progress of these reactions and to assess the nature of resulting oligomeric products.

A novel polyamine acyltransferase responsible for the accumulation of spermidine conjugates in Arabidopsis seed.

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4'-O-glycosyl)-sinapoyl),N'-sinapoylspermidine and N,N'-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.

Organocatalytic aziridine synthesis using F+ salts.

This paper describes a unique application of the fluoronium cation (F+) as an organocatalyst for mediating the reaction between N-substituted imines and ethyl diazoacetate affording excellent yields of N-substituted aziridines.

Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide.

Cytochrome cd(1) is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd(1) is mixed with nitrite in a stopped-flow apparatus in the absence of excess reductant, a kinetically stable complex of enzyme and product forms, assigned as a mixture of cFe(II) d(1)Fe(II)-NO(+) and cFe(III) d(1)Fe(II)-NO (cd(1)-X). However, in order for the enzyme to achieve steady-state turnover, product (NO) release must occur. In this work, we have investigated the effect of a physiological electron donor to cytochrome cd(1), the copper protein pseudoazurin, on the mechanism of nitrite reduction by the enzyme. Our data clearly show that initially oxidized pseudoazurin causes rapid further turnover by the enzyme to give a final product that we assign as all-ferric cytochrome cd(1) with nitrite bound to the d(1) heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd(1). In contrast, redox-inert zinc pseudoazurin did not affect cd(1)-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd(1)-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd(1)-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd(1) functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone.

Analysis of the nitric oxide-sensing non-heme iron center in the NorR regulatory protein.

The NorR regulatory protein senses nitric oxide (NO) to activate genes required for NO detoxification under anaerobic and microaerobic conditions in Escherichia coli. NorR belongs to the sigma(54)-dependent family of transcriptional activators and contains an N-terminal regulatory GAF (cGMP phosphodiesterase, adenylate cyclase, FhlA) domain that controls the ATPase activity of the central AAA+ domain to regulate productive interactions with sigma(54). Binding of NO to a non-heme iron center in the GAF domain results in the formation of a mononitrosyl-iron complex and releases intramolecular repression of the AAA+ domain to enable activation of transcription. In this study, we have further characterized NorR spectroscopically and substituted conserved residues in the GAF domain. This analysis, in combination with structural modeling of the GAF domain, has identified five candidate ligands to the non-heme iron and suggests a model in which the metal ion is coordinated in a pseudo-octahedral environment by three aspartate residues, an arginine, and a cysteine.