Anti-hyperuricemia activity and toxicity prediction of a novel xanthine oxidoreductase inhibitor

A potent xanthine oxidoreductase inhibitor (LS087) was recently proved to exhibit a similar hypouricemic potency to febuxostat. A hyperuricemia model induced by potassium oxonate and hypoxanthine was proposed in specific pathogen-free male Kunming mice, and the serum urea nitrogen, creatinine and uric acid levels were measured after oral administration of LS087. Furthermore, renal histopathology was conducted by staining with hematoxylin and eosin, periodic acid–Schiff and Masson's trichrome stains, respectively. The results showed that the levels of serum urea nitrogen and uric acid significantly decreased compared with the model group, but the level of creatinine showed no significant changes. The pathological abnormalities in kidney tubules were improved after LS087 administration. Ten metabolites (M1–M10) of LS087 were identified after a single oral dosing of 10 mg/kg in rats. M6 was the primary LS087 metabolite in vivo with a pathway of methylation. The toxicity and potential risks of LS087 and its metabolites were predicted using the ProTox-II software. LS087 and the major metabolites (M2, M3, M5, M6, M7 and M8) were predicted to have no potential hepatotoxicity, but some metabolites with a total rate of <1% (M1, M4, M9, and M10) showed potential hepatotoxicity. M1 and M8 showed potential carcinogenicity. The LS087 biotransformation pathway in rat was well characterized.     

Surface energy modified chips for detection of conformational states and enzymatic activity in biomolecules

A novel patterning method for anchoring biomolecules and noncovalent assembled conjugated polyelectrolyte (CPE)/biomolecule complexes to a chip surface is presented. The surface energy of a hydrophilic substrate is modified using an elastomeric poly(dimethylsiloxane) (PDMS) stamp, containing a relief pattern. Modification takes place on the parts where the PDMS stamp is in conformal contact with the substrate and leaves low molecular weight PDMS residues on the surface resulting in a hydrophobic modification, and then biomolecules and CPE/biomolecule complexes are then adsorbed in a specific pattern. The method constitutes a discrimination system for different conformations in biomolecules using CPEs as reporters and the PDMS modified substrates as the discriminator. Detection of different conformations in two biomacromolecules, a synthetic peptide (JR2E) and a protein (calmodulin), reported by the CPE and resolved by fluorescence was demonstrated. Also, excellent enzyme activity in patterned CPE/horseradish peroxidase (HRP) enzyme was shown, demonstrating that this method can be used to pattern biomolecules with their activity retained. The method presented could be useful in various biochip applications, such as analyzing proteins and peptides in large-scale production, in making metabolic chips, and for making multi-microarrays.     

Protein film voltammetry of Rhodobacter capsulatus xanthine dehydrogenase

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD(+) as the electron acceptor. R. capsulatus XDH forms an (alphabeta)(2) heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds; however, R.capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinant enzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite working electrode. Furthermore, we have determined all redox potentials of the four cofactors (Mo(VI/V), Mo(V/IV), FAD/FADH, FADH/FADH(2) and two distinct [2Fe-2S](2+/+) clusters) using a combination of potentiometric and voltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potential for the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis.     

Protein conformational switches: from nature to design

Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation--which can be carried out by a molecule as small as a thousand atoms or so--belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, biotechnology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and "smart" biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding-unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.     

Oxidative coupling of epigallocatechin gallate amplifies antioxidant activity and inhibits xanthine oxidase activity

Oxidative coupling of epigallocatechin gallate resulted in great improvement in antioxidant activity such as radical scavenging activity against superoxide anion and in activity to inhibit xanthine oxidase, offering high potential as a therapeutic agent for prevention of xanthine oxidase-induced diseases such as gout.     

A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(?)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 ? between the two subunits by a specific pathway (Y(122)(?)→W(48)→Y(356) within β to Y(731)→Y(730)→C(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(?)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-β2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(?) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(?)/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(?) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(?) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(?) is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-β2, a mechanism for PCET uncoupling in NO(2)Y(?)-RNR is proposed.     

A biomimetic approach to oxidized sites in the xanthine oxidoreductase family: synthesis and stereochemistry of tungsten(VI) analogue complexes

Two series of square pyramidal (SP) monodithiolene complexes, [M (VI)O 3- n S n (bdt)] (2-) and their silylated derivatives [M (VI)O 2- n S n (OSiR 3)(bdt)] (-) ( n = 0, M = Mo or W; n = 1, 2, M = W), synthesized in this and previous work, constitute the basic molecules in a biomimetic approach to structural analogues of the oxidized sites in the xanthine oxidoreductase enzyme family. Benzene-1,2-dithiolate (bdt) simulates native pyranopterindithiolene chelation in the basal plane, tungsten instead of the native metal molybdenum was employed in sulfido complexes to avoid autoreduction, and silylation models protonation. The complexes [MO 3(bdt)] (2-) and [MO 2(OSiR 3)(bdt)] (-) represent inactive sites, while [MO 2S(bdt)] (2-) and [MOS(OSiR 3)(bdt)] (-), with basal sulfido and silyloxo ligands, are the first analogues of the catalytic sites. Also prepared were [MOS 2(bdt)] (2-) and [MS 2(OSiR 3)(bdt)] (-), with basal sulfido and silyloxo ligands. Complexes are described by angular parameters which reveal occasional distortions from idealized SP toward a trigonal bipyramidal (TBP) structure arising from crystal packing forces in crystalline Et 4N (+) salts. Miminized energy structures from DFT calculations are uniformly SP and reproduce experimental structures. For example, the correct structure is predicted for [WO 2S(bdt)] (2-), whose basal and apical sulfido diastereomers are potentially interconvertible through a low-lying TBP transition state for pseudorotation. The lowest energy tautomer of the protonated form is calculated to be [WOS(OH)(bdt)] (-), with basal sulfido and hydroxo ligands. Computational and experimental structures indicate that protein sites adopt intrinsic coordination geometries rather than those dictated by protein structure and environment.     

Specific anion effects on enzymatic activity in nonaqueous media

The present work shows that salt anions affect the activity of Pseudomonas cepacia lipase both in aqueous and in nonaqueous media (NAM) according to a Hofmeister series. The biocatalytic assay in water was the hydrolysis of p-nitrophenyl acetate, whereas the esterification between 1-hexyl-beta-D-galactopyranoside and palmitic acid was followed in an organic solvent. The solid lipase preparations to be used in NAM were obtained through lyophilization in the presence of concentrated solutions of Hofmeister salts (Na2SO4, NaH2PO4/Na2HPO4, NaCl, NaBr, NaI, NaSCN). Salts affect enzyme activity in organic media through two mechanisms: (1) enzyme protection during lyophilization; (2) enzyme activation during the reaction. At least in our case, the latter seems to be more important than the former. The decrease of the activation energy caused by the stabilization of the transition state due to "kosmotropic" anions might be the driving force of enzyme activation. According to the most recent findings, dispersion forces may be responsible of specific anion enzyme activation/deactivation in NAM.     

Charge-transfer mechanism for cytochrome c adsorbed on nanometer thick films. Distinguishing frictional control from conformational gating

Using nanometer thick tunneling barriers with specifically attached cytochrome c, the electron-transfer rate constant was studied as a function of the SAM composition (alkane versus terthiophene), the omega-terminating group type (pyridine, imidazole, nitrile), and the solution viscosity. At large electrode-reactant separations, the pyridine terminated alkanethiols exhibit an exponential decline of the rate constant with increasing electron-transfer distance. At short separations, a plateau behavior, analogous to systems involving -COOH terminal groups to which cytochrome c can be attached electrostatically, is observed. The dependence of the rate constant in the plateau region on system properties is investigated. The rate constant is insensitive to the mode of attachment to the surface but displays a significant viscosity dependence, change with spacer composition (alkane versus terthiophene), and nature of the solvent (H(2)O versus D(2)O). Based on these findings and others, the conclusion is drawn that the charge-transfer rate constant at short distance is determined by polarization relaxation processes in the structure, rather than the electron tunneling probability or large-amplitude conformational rearrangement (gating). The transition in reaction mechanism with distance reflects a gradual transition between the tunneling and frictional mechanisms. This conclusion is consistent with data from a number of other sources as well.     

Fluorescence activated cell sorting for enzymatic activity

Directed evolution is a reliable method for protein engineering and as a tool for investigating structure/function relationships. A key for a successful directed evolution experiment is oftentimes the screen. Fluorescence activated cell sorting (FACS) is powerful high-throughput screening approach to isolate and identify mutants from large protein libraries. FACS has been successful in isolating proteins with improved or altered binding affinity. However, FACS screening for mutants with enhanced catalytic activity has been met with limited success. This review focuses on the FACS screening of protein libraries for enzymatic activity.     

Stereoselective enzymatic synthesis of monoglucosyl-myo-inositols with in vivo anti-inflammatory activity

The monoglucosyl-inositols α-d-glucopyranosyl-(1→4)-4d-myo-inositol 3 and α-d-glucopyranosyl-(1→1)-1d-myo-inositol 4 were synthesized by a combined enzymatic transglucosylation and hydrolysis strategy, using cyclodextrin glucosyl transferase (CGTase) from Thermoanaerobacter sp., followed by hydrolysis with Aspergillus niger glucoamylase. The glucosides were separated by preparative HPLC and fully characterized by extensive 1D and 2D NMR studies. The structure of the regioisomer 4 was confirmed by X-ray crystallography of its perbenzoylated derivative 4a. Both isomers demonstrated in vivo anti-inflammatory activity at comparative levels to corticosterone on mouse ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and in rat hind paw oedema induced by carrageenan.     

Amperometric detection of hypoxanthine and xanthine by enzymatic amplification using a gold nanoparticles-carbon nanohorn hybrid as the carrier

A novel gold nanoparticles-single-walled carbon nanohorn (GNPs-SWCNH) hybrid was synthesized for the construction of an amperometric biosensing platform. The GNPs-SWCNH hybrid was stable in aqueous solution for at least two weeks, and was characterized with scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy. The average diameter of GNPs in situ synthesized on the SWCNH was 5-8 nm, and the good interaction between GNPs and SWCNH was confirmed by ultraviolet-visible absorption spectroscopy. The GNPs-SWCNH immobilized on a platinum electrode showed high electrochemical activity toward the oxidation of hydrogen peroxide and uric acid with low applied potentials. Combining with the enzymatic reaction of xanthine oxidase (XOx), a biosensor for hypoxanthine and xanthine was constructed. The XOx-GNPs-SWCNH-based biosensor exhibited good responses to hypoxanthine and xanthine with the linear ranges of 1.5 to 35.4 and 2.0 to 37.3 μM, and the detection limits of 0.61 and 0.72 μM, respectively. The recovery test showed acceptable results. The gold nanoparticles functionalized carbon nanohorns provided a promising way to construct an electrochemical platform for sensitive biosensing.     

DPPH radical scavenging and xanthine oxidase inhibitory activity of Terminalia macroptera leaves

From a methanol extract of the leaves of the Malian medicinal tree Terminalia macroptera, cis-polyisoprene (1), chebulic acid trimethyl ester (2), methyl gallate (3), shikimic acid (4), corilagin (5), rutin (6), narcissin (7), chebulagic acid (8) and chebulinic acid (9), were isolated. Cispolyisoprene (1) was the major non-polar constituent. The novel compound 2 showed high radical scavenging activity (IC50 4.7 microg/mL), but was inactive as xanthine oxidase inhibitor. The major substituent of the crude extract, substance 5, showed a high radical scavenger effect (IC50 2.7 microg/mL) and weak xanthine oxidase inhibition (IC50 ca 105 microg/mL). The antioxidant and radical scavenging effects of some of the substances identified in this study may to some extent explain the medical use of this tree in West Africa.     

Preparation of ferulic acid derivatives and evaluation of their xanthine oxidase inhibition activity

Several ferulic acid ethyl esters (3a-h) were synthesized under the Knoevengel reaction condition and they were further reduced to afford the respective allylic alcohol derivatives (4a-g). Some of them were evaluated for the xanthine oxidase (XO) inhibitory activity. Among them, 3h exhibited a significant inhibitory activity with an IC50 value of 1.35 x 10(-5) M, while the IC50 value of allopurinol used as the positive control was 1.49 x 10(-5) M. The study suggested that the higher acidity of the phenolic OH group in the ferulic acid derivatives might result in improved XO inhibitory activity.     

A new mathematical formula to link near equilibrium relaxation kinetics and conformational selection steps in enzymatic reactions

A new mathematical formula was derived for near equilibrium relaxation processes of enzyme reactions including the conformational selection (CS) modes. CS is one of the most accepted molecular recognition mechanisms, in which protein conformers (CS conformers) are in an equilibrium with varying degrees of ligand binding affinity so that ligands select a particular conformer among them to bind. Using computer simulation techniques, our previous study (Egawa and Callender in Math Biosci 313: 61–70, 2019) predicted that the rate constant for the near equilibrium relaxation processes (k_NER) and the concentration-sum of substrate and product (C_L^t) of enzyme reactions uniquely related to the presence of CS steps in a manner that 1/k_NER versus C_L^t plot transformed from linear to quadratic as the elementary rate constants of inter-conversions among the CS conformers were becoming smaller relative to the rate constants at other steps of the enzymatic reaction system. Thus our previous work could provide a potential tool to detect the presence of CS steps in an enzyme reaction simply by assays using only trace amount of enzyme samples, although logical basis to have the quadratic deformation in the 1/k_NER versus C_L^t plot in response to the presence of CS steps could not be clarified. Employing mathematical approaches that were alternative to those in our previous study, this study succeeded in deriving a theoretical equation that fully explained why and how the CS modes caused the quadratic characters in the 1/k_NER versus C_L^t plot.

     

Chemical synthesis of human selenoprotein F and elucidation of its thiol-disulfide oxidoreductase activity

Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl₂, thus facilitating the synthesis of multi-milligrams of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.

The chemical synthesis of the 134-residue human selenoprotein F (SelF) was accomplished on a multi-milligram scale. The synthetic SelF exhibits typical thiol-disulfide oxidoreductase activity.     

Multi-walled carbon nanotubes decrease lactate dehydrogenase activity in enzymatic reaction

The oxidation of 1, 4-nicotinamide adenine dinucleotide (NADH) to β-nicotinamide adenine dinucleotide (NAD(+)) coupled with converting of pyruvic acid (PA) to lactate catalyzed by lactate dehydrogenase (LDH), NADH+PA+H(+)?LDHNAD(+)+Lactate, was widely adopted to quantify the cell's death, membrane infiltration and proliferation induced by potential toxins. The differential pulse voltammetry (DPV) cathodic signal of NAD(+) at a hanging mercury drop electrode (HMDE) showed LDH activity decreased with the elevating dosages of and the pre-contact time (t(c)) with multi-walled carbon nanotubes (MWCNTs). Comparison of kinetic rate constant of above enzymatic reaction (ER) was able to sensitively assay the adverse influence of MWCNTs. Toxic concentration of altering relative LDH activity by 50% (TC(50)) of MWCNTs was derived to be 40mg/L. TC(50) values indicated a decrease toxicity order Al (III)>MWCNTs>nano-Al(13)>50nm-Al(2)O(3)≥1000nm-Al(2)O(3). The negatively charged surfaces of these nanoparticles (NPs) might be a main cause for the decrement of LDH activity. This decrement was capable to result in the underestimation of the toxicity of NPs in classic LDH assays. This observation would highlight to settle down contradictory medium dependent toxicity of MWCNTs among the literature.