Immobilized respiratory chain activities fromEscherichia coli utilized to measure d and l-lactate,succinate, l-malate, 3-glycerophosphate,pyruvate, or NAD(P)H

The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.     

Direct electrochemical interaction between a modified gold electrode and a bacterial membrane extract

A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.     

cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer.

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.     

Investigating the Thermostability of Succinate: Quinone Oxidoreductase Enzymes by Direct Electrochemistry at SWNTs‐Modified Electrodes and FTIR Spectroscopy

Succinate: quinone reductases (SQRs) are the enzymes that couple the oxidation of succinate and the reduction of quinones in the respiratory chain of prokaryotes and eukaryotes. Herein, we compare the temperature‐dependent activity and structural stability of two SQRs, the first from the mesophilic bacterium Escherichia coli and the second from the thermophilic bacterium Thermus thermophilus, using a combined electrochemical and infrared spectroscopy approach. Direct electron transfer was achieved with full membrane protein complexes at single‐walled carbon nanotube (SWNT)‐modified electrodes. The possible structural factors that contribute to the temperature‐dependent activity of the enzymes and, in particular, to the thermostability of the Thermus thermophilus SQR are discussed.     

On the Importance of Anion–π Interactions in the Mechanism of Sulfide:Quinone Oxidoreductase

Sulfide:quinone oxidoreductase (SQR) is a flavin‐dependent enzyme that plays a physiological role in two important processes. First, it is responsible for sulfide detoxification by oxidizing sulfide ions (S^2? and HS^?) to elementary sulfur and the electrons are first transferred to flavin adenine dinucleotide (FAD), which in turn passes them to the quinone pool in the membrane. Second, in sulfidotrophic bacteria, SQRs play a key role in the sulfide‐dependent respiration and anaerobic photosynthesis, deriving energy for their growth from reduced sulfur. Two mechanisms of action for SQR have been proposed: first, nucleophilic attack of a Cys residue on the C4 of FAD, and second, an alternate anionic radical mechanism by direct electron transfer from Cys to the isoalloxazine ring of FAD. Both mechanisms involve a common anionic intermediate that it is stabilized by a relevant anion–π interaction and its previous formation (from HS^? and Cys‐S‐S‐Cys) is also facilitated by reducing the transition‐state barrier, owing to an interaction that involves the π system of FAD. By analyzing the X‐ray structures of SQRs available in the Protein Data Bank (PDB) and using DFT calculations, we demonstrate the relevance of the anion–π interaction in the enzymatic mechanism.     

Synthesis, structural analysis, and magnetic behaviour of three fumarate bridged coordination polymers: five-fold interpenetrated diamond-like net of NiII, sheets of NiII and CoII

The hydrothermal reactions of Ni(NO(3))(2).6H(2)O, disodium fumarate (fum) and 1,2-bis(4-pyridyl)ethane (bpe)/1,3-bis(4-pyridyl)propane (bpp) in aqueous-methanol medium yield one 3-D and one 2-D metal-organic hybrid material, [Ni(fum)(bpe)] (1) and [Ni(fum)(bpp)(H(2)O)] (2), respectively. Complex possesses a novel unprecedented structure, the first example of an "unusual mode" of a five-fold distorted interpenetrated network with metal-ligand linkages where the four six-membered windows in each adamantane-type cage are different. The structural characterization of complex 2 evidences a buckled sheet where nickel ions are in a distorted octahedral geometry, with two carboxylic groups, one acting as a bis-chelate, the other as a bis-monodentate ligand. The metal ion completes the coordination sphere through one water molecule and two bpp nitrogens in cis position. Variable-temperature magnetic measurements of complexes 1 and 2 reveal the existence of very weak antiferromagnetic intramolecular interactions and/or the presence of single-ion zero field splitting (D) of isolated Ni(II) ions in both the compounds. Experimentally, both the J parameters are close, comparable and very small. Considering zero-field splitting of Ni(II), the calculated D values are in agreement with values reported in the literature for Ni(II) ions. Complex 3, [[Co(phen)]](2)(fum)(2)](phen = 1,10-phenanthroline) is obtained by diffusing methanolic solution of 1,10-phenanthroline on an aqueous layer of disodium fumarate and Co(NO(3))(2).6H(2)O. It consists of dimeric Co(II)(phen) units, doubly bridged by carboxylate groups in a distorted syn-syn fashion. These fumarate anions act as bis-chelates to form corrugated sheets. The 2D layer has a (4,4) topology, with the nodes represented by the centres of the dimers. The magnetic data were fitted ignoring the very weak coupling through the fumarate pathway and using a dimer model.     

Structure Elucidation of a Complex Faldaprevir‐(±)‐α‐Tocopherol Addition Product Reveals Reactivity of Common Excipient Vitamin E‐TPGS

Vitamin E‐TPGS (d ‐α‐tocopheryl polyethylene glycol succinate) is a common excipient used in drug formulations. This excipient is formed by esterification of Vitamin E succinate with polyethylene glycol. As part of an oral formulation for active pharmaceutical ingredient (API) Faldaprevir, Vitamin E‐TPGS was found to decompose under ambient conditions producing free (±)‐α‐Tocopherol which subsequently formed an adduct composed of α‐Tocopherol and the API. The addition product was isolated using liquid chromatography with collections onto solid‐phase extraction cartridges, and full structure elucidation was achieved using mass spectrometry and nuclear magnetic resonance spectroscopy. The results revealed a regioselective addition of α‐Tocopherol to the API that likely occurs through the formation of a stabilized ortho‐quinone methide intermediate. This finding demonstrates the propensity of the common excipient (Vitamin E‐TPGS) to generate chemically active intermediates that may react with formulation ingredients.     

Inhibitory effects of galloylglucose on nicotinamide adenine dinucleotide dehydrogenases of the aerobic respiratory chain of Escherichia coli

The effects of pentagalloylglucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucose) on the aerobic electron transport system of Escherichia coli were studied. The activity of nicotineamide adenine dinucleotide (NADH) reductase was inhibited by pentagalloylglucose, but the activities of succinate dehydrogenase, D-lactate dehydrogenase, and ubiquinol-1 (Q1H2) oxidase were not susceptible to the inhibitor. Because the presence of two kinds of NADH dehydrogenase in respiratory chain of Escherichia coli has been reported, we examined the effect of galloylglucose independently on both NADH dehydrogenases. Pentagalloylglucose is potent and specific inhibitor of both NADH dehydrogenases. One of the NADH dehydrogenases (NADH dh II) is more sensitive to the inhibitor than the other (NADH dh I).     

Bioconversion of fumarate to succinate using glycerol as a carbon source

In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated. The yield of biocon version was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L. When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration. The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L. Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture. This strain converted fumarate to succinate at a rate of 3.5 g/L·h under the sparge of CO₂.     

Bioelectrocatalysts

Fumarate reductase was used as a model oxidoreductase to demonstrate continuous electrical cofactor reduction-oxidation during the bioelectrochemical synthesis and detection of chemicals. The enzyme preparation was immobilized onto a graphite felt electrode that was modified with carboxymethylcellulose (CMC). Nicotinamide adenine dinucleotide (NAD), neutral red, and fumarate reductase (which contained menaquinone) were covalently linked by peptide bonds to the CMC. The electron mediator neutral red allowed NAD and menaquinone to be recycled electrically during enzymatic chemical synthesis. Succinate detection by the bioelectrocatalyst was linear from 5 μM to 10 mM succinate. Fumarate synthesis using this bioelectrode was dependent on succinate utilization and resulted in proportional production of electricity and fumarate. Succinate synthesis using this bioelectrocatalyst was dependent on current and fumarate concentration. This bioelectrocatalyst system may enhance the utility of menaquinone- and/or pyridine nucleotide-linked oxidoreductases in diverse enzymatic fuel cells and sensors. It may also enhance the utility of oxidoreductase-based chemical synthesis systems because it eliminates the problem of cofactor recycling.     

Construction of Hybrid Enzyme from HEC-SOD and Cu,Zn-SOD

Human extracellular superoxide dismutase(hEC-SOD) is a secreted tetrameric protein involved in the protection of a human body from oxygen free radicals. Its three-dimensional structure has not been confirmed, hEC-SOD couldn′t be expressed in E.coll. We constructed a hybrid enzyme, which comprises the Nterminal and C-terminal domains from hEC-SOD, fused it to human Cu, Zn-SOD. The hybrid enzyme is expressed successfully in E. coli. Further, we analyzed the expression of hEC-SOD in E. coli by mRNA differential displaying.     

Continuous production of succinic acid by a fumarate-reducing bacterium immobilized in a hollow-fiber bioreactor

Enterococcus faecalis RKY1, a fumarate-reducing bacterium, was immobilized in an asymmetric hollow-fiber bioreactor (HFBR) for the continuous production of succinic acid. The cells were inoculated into the shell side of the HFBR, which was operated in transverse mode. Since the pH values in the HFBR declined during continuous operation to about 5.7, it was necessary to change the feed pH from 7.0 to 8.0 after 24 h of operation in order to enhance production of succinic acid. During continuous operation with a medium containing fumarate and glycerol, the productivity of succinate was 3.0–10.9 g/(L·h) with an initial concentration of 30 g/L of fumarate, 4.9–14.9 g/(L·h) with 50 g/L of fumarate, and 7.2–17.1 g/(L·h) with 80 g/L of fumarate for dilution rates between 0.1 and 0.4 h⁻¹. The maximum productivity of succinate obtained by the HFBR (17.1 g of succinate /[L·h]) was 1.7 times higher than that of the batch bioconversions (9.9 g of succinate /[L·h]) with 80 g/L of fumarate. Furthermore, the long-term stability of the HFBR was demonstrated with a continuously efficient production of succinate for more than 15 d (360 h).     

An analysis of the effect of crosslinking on the Tg of polyester and other networks

The dynamic mechanical T_g of styrene-poly(diethylene succinate fumarate) networks has been quantitatively related to the crosslink density. The crosslinking constants, derived from this analysis, were compared with those obtained from other network systems. It is concluded that the crosslinking constant of Di Marzio is not a universal constant; however, a related parameter was found to be very nearly material independent.     

Influence of the carbon-carbon double bond on the coordinative ability of unsaturated bicarboxylic ligands in aqueous solution. A thermodynamic study

Enthalpies of complex formation of fumarate, itaconate, maleate and succinate with calcium have been determined by direct calorimetry. By using the stability constants of the literature, Gibbs function and entropy were also obtained. The present data refer to the aqueous medium at T = 25°C and I = 0.5 mol l^?1. In spite of the trans-configuration of fumarate, examination of the present thermodynamic results indicates a probable bidentate behaviour for this ligand, as a consequence of a charge-transmission due to the carbon-carbon double bond. The lower stability of the fumarate complexes in comparison with those of its cis-isomer seems due to different solute-solvent interactions, while the metalligand bond strength seems similar in both cases. On the other hand the present results seem to indicate for the itaconate complexes a structure similar to that of maleate and succinate, without charge transmission.     

Manganese(II) zero-field interaction in cambialistic and manganese superoxide dismutases and its relationship to the structure of the metal binding site

The Mn(II) high-magnetic-field electron paramagnetic resonance (HFEPR) spectra of five different superoxide dismutases (SODs) were measured at 190 and 285 GHz. The native E. coli manganese SOD was found to be distinct from the other SODs by virtue of its large zero-field E-value. The two wild-type cambialistic proteins from Porphyromonas gingivalis and Rhodobacter capsulatus were also distinct. However, the Gly155Thr mutant of the P. gingivalis SOD changed the Mn(II) spectrum so that it closely resembled the spectrum of manganese reconstituted E. coli iron SOD. This observation paralleled enzyme activity measurements that show that this mutation causes the loss of activity with manganese and enhanced activity with iron indicating a conversion from a cambialistic to an iron-specific protein. The Mn(II) magnetic parameters were determined by simultaneously fitting the multifrequency data. Simulations were carried out by numerically diagonalizing the spin Hamiltonian and explicitly calculating all possible transition probabilities. The relationship between the Mn(II) zero-field interaction and structure of the metal binding site is also discussed.     

Synthesis and characterization of biodegradable poly(butylene succinate-co-butylene fumarate)s

A series of aliphatic biodegradable polyesters modified with fumaric residues was synthesized by transesterification in the melt of dimethyl succinate, dimethyl fumarate and 1,4-butanediol. The amount of unsaturation, originating from the fumaric acid residues in the polyesters chains was varied from 5 to 20 mol%. The molecular structure and composition of the polyesters were determined by ¹H NMR spectroscopy. The effects of the content of fumaric residues on the thermal and thermo-oxidative properties of the synthesized polyesters were investigated using differential scanning calorimetry (DSC) and thermogravimetric analysis. The degree of crystallinity was determined by DSC and wide angle X-ray scattering. The degrees of crystallinity of the unsaturated copolyesters were reduced, while the melting temperatures were higher in comparison to poly(butylene succinate). Biodegradation of the synthesized copolyesters was estimated in enzymatic degradation tests using a buffer solution with Rhizopus arrhizus lipase at 37 °C. Although the degree of crystallinity of the copolyesters decreases slightly with increasing unsaturation, the biodegradation is not enhanced suggesting that not only the chemical structure and molecular stiffness but also the morphology of the spherulites has an influence on the biodegradation properties. The highest biodegradability was observed for the copolyesters containing 5 and 10 mol% of fumarate units.     

The PcoC copper resistance protein coordinates Cu(I) via novel S-methionine interactions

The E. coli copper resistance protein PcoC enhances survival of a bacterium under conditions of extreme copper stress. This small protein has no cysteines, but does have an unusual methionine-rich sequence motif, suggesting that methionine ligation may be important in Cu binding. It is shown that PcoC binds both Cu(I) and Cu(II), in addition to binding Hg(II) and Ag(I). Previously crystallographic studies of PcoC had shown that the apo protein adopts a beta-barrel fold typical of that seen for blue-copper electron-transfer proteins. However, in contrast with electron-transfer proteins, where the Cu(I) and Cu(II) structures are nearly identical, X-ray absorption spectra show that the structures of the Cu site in reduced and oxidized PcoC are dramatically different. Cu(II) PcoC has a tetragonal Cu structure in which the Cu is coordinated to O or N ligands, including at least two histidine ligands. Cu(I) PcoC has a trigonal site with two methionine ligands. This is the first well-characterized example of a methionine-rich protein Cu binding site, demonstrating a new type of biological Cu coordination chemistry.     

One-dimensional zinc(II) fumarate coordination polymers

A new procedure developed for the synthesis and crystallization of various zinc(II) fumarate hydrate coordination polymers is described. In the first step, anhydrous Zn(II) fumarate, [Zn(C₄H₂O₄)] (1), is synthesized from Zn(II) acetate and fumaric acid in methanol. Subsequently, this product is used as a starting material for growing small crystals of bis–aqua Zn(II) fumarate, [Zn(H₂O)₂(C₄H₂O₄)] (2), triaqua Zn(II) fumarate monohydrate, [Zn(H₂O)₃(C₄H₂O₄)]·H₂O (3), tetraaqua Zn(II) fumarate, [Zn(H₂O)₄(C₄H₂O₄)] (4), and tetraaqua Zn(II) fumarate monohydrate, [Zn(H₂O)₄(C₄H₂O₄)]·H₂O (5). All structures were determined or redetermined by X-ray structure analyses. The hitherto unknown compound 3 exhibits a zig-zag chain structure with five-coordinate Zn(II) ions.     

Structure-activity relationships of galabioside derivatives as inhibitors of E. coli and S. suis adhesins: nanomolar inhibitors of S. suis adhesins

Four collections of Gal alpha1-4Gal derivatives were synthesised and evaluated as inhibitors of the PapG class II adhesin of uropathogenic Escherichia coli and of the P(N) and P(O) adhesins of Streptococcus suis strains. Galabiosides carrying aromatic structures at C1, methoxyphenyl O-galabiosides in particular, were identified as potent inhibitors of the PapG adhesin. Phenylurea derivatisation at C3' and methoxymethylation at O2' of galabiose provided inhibitors of the S. suis strains type P(N) adhesin with remarkably high affinities (30 and 50 nM, respectively). In addition, quantitative structure-activity relationship models for E. coli PapG adhesin and S. suis adhesin type P(O) were developed using multivariate data analysis. The inhibitory lead structures constitute an advancement towards high-affinity inhibitors as potential anti-adhesion therapeutic agents targeting bacterial infections.     

An efficient method for synthesis of succinate-based MMP inhibitors

A differentially protected fumarate undergoes radical addition followed by allylstannane trapping to provide disubstituted succinates in good yields and high anti diastereoselectivity. The conversion of the succinate to a known MMP inhibitor has been accomplished. [reaction: see text]