Goedele Roos

Vrije Universiteit Brussel | VUB

The arginine-agmatine antiporter (AdiC) is a component of an acid resistance system developed by enteric bacteria to resist gastric acidity. In order to avoid neutral proton antiport, the monovalent form of arginine, about as abundant as its divalent form under acidic conditions, should be selectively bound by AdiC for transport into the cytosol. I...

Located at the tip of type I fimbria of Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibiti...

Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: a) weakening of the leaving- group bond, thereby decreasing activation strain; and b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrat...

Biophysical techniques are used in many key stages of the drug discovery process including in screening for new receptor ligands, in characterising drug mechanisms, and in validating data from biochemical and cellular assays. This book provides an overview of the biophysical methods applied in drug discovery today, including traditional techniques...

The most prevalent diseases manifested by it Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease. it E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected it via a pilin domain to the tip of type 1 pili. Although the is...

Recently, we extended the anti-adhesive concept showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogs of heptylmannoside (HM), a previously identified nanomolar FimH inhibitor, but...

Atomic charges are a key concept to give more insight into the electronic structure and chemical reactivity. The Hirshfeld-I partitioning scheme applied to the model protein human 2-cysteine peroxiredoxin thioredoxin peroxidase B is used to investigate how large a protein fragment needs to be in order to achieve convergence of the atomic charge of...

Just like bacteria need to be mobile to seek for nutrients, bacteria need to adhere to biotic and abiotic surfaces to enable their progression. Most bacteria regulate the expression of a multitude of fimbrial adhesins that display varying specificities and architectures. FimH at the tip of type 1 fimbriae is one of the first recognized lectins on E...

Hydrogen peroxide is a natural oxidant that can oxidize protein thiols (\ce{RSH}) via sulfenic acid (\ce{RSOH}) and sulfinic acid (\ce{RSO2H}) to sulfonic acid (\ce{RSO3H}). In this paper, we study the complete anionic and neutral oxidation pathway from thiol to sulfonic acid. Reaction barriers and reaction free energies for all three oxidation ste...

Protein thiol/sulfenic acid oxidation potentials provide a tool to select specific oxidation agents, but are experimentally difficult to obtain. Here, insights into the thiol sulfenylation thermodynamics are obtained from model calculations on small systems and from a quantum mechanics/molecular mechanics (QM/MM) analysis on human 2-Cys peroxiredox...

Adherent-Invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with CD, adhere to and invade intestinal epithelial cells with Crohn’s disease (CD). We developed a set of mannosides to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. The crystal structure of the FimH lectin domain in complex with a l...

Protein disulfides can adopt a wide variety of conformations, each having different energies. Limited experimental data suggest that disulfides adopting a high energy have an enhanced likelihood for reduction, but the exact nature of this relation is not clear. Using a computational approach, we give insight on the conformational dependence of the...

Antagonists of the FimH adhesin, a protein almost universally present at the extremity of type-1 fimbriae expressed by Escherichia coli, have been abundantly in the spotlight as alternative treatments of urinary tract infections. The antagonists function as bacterial antiadhesives through highly specific α-D-mannose binding in a charged and polar p...

doi:10.1038/scibx.2013.644

Adherent-Invasive Escherichia coli (AIEC) have previously been shown to induce gut inflammation in patients with Crohn?s disease (CD). We developed a set of mannosides to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. The crystal structure of the FimH lectin domain in complex with a lead thiazolylaminomannoside highlight...

The effect of non-polar and polar ligands and of monovalent cations on the one-electron reduction potential of the thiyl radical and the disulfide bond was evaluated. The reduction potentials E° for the CH(3) S(.) -n L/CH(3) S(-) -n L and CH(3) SSCH(3) -L/CH(3) SSCH(3) (.) (-) -L redox couples were calculated at the B3LYP, M06-2X and MP2 levels of...

To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) wi...

Abstract Many cellular functions involve cysteine chemistry via thiol-disulfide exchange pathways. The nucleophilic cysteines of the enzymes involved are activated as thiolate. A thiolate is much more reactive than a neutral thiol. Therefore, determining and understanding the pK(a)s of functional cysteines are important aspects of biochemistry and...

Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, because of their highly reactive nature. We designed a calculation method, the reduction potentials from electron...

Protein sulfenic acid formation has long been regarded as unwanted damage caused by reactive oxygen species (ROS). However, over the past 10 years, accumulating evidence has shown that the reversible oxidation of cysteine thiol groups to sulfenic acid functions as a redox-based signal transduction mechanism. Here, we review the mechanisms of sulfen...

Author Summary Thioredoxins are found in all types of cells and control several essential functions of life, including promotion of cell growth, inhibition of apoptosis, and modulation of inflammation. Thioredoxin has two ‘free’ cysteines in its active site, which are used to break disulfide bonds in oxidized substrate proteins. In the first step,...

Experimentalists and quantum chemists are living in a different world. A wealth of theoretical enzymology-related publications is hardly known by experimentalists, and vice versa. Our aim is to bring both worlds together and to show the powerful possibilities of a multidisciplinary approach to study subtle details of complicated enzymatic processes...

Models of the Bs_Trx, Bs_ArsC and B. subtilis Trx-ArsC complex for DFT calculations (0.03 MB DOC)

Hydrogen bonds formed with Arg16 (0.38 MB DOC)

Description of reactivity (0.03 MB DOC)

Site-directed mutagenesis, expression and purification of Sa_Trx and Sa_ArsC (0.03 MB DOC)

Model system of the Trx systems (7.08 MB TIF)

Determination of the pKa of Cys89 of Sa_ArsC C10S/C15A/C82A and of Cys10 of oxidized Sa_ArsC C15A (0.10 MB DOC)

Calculated and experimentally obtained pKas of different Trx and ArsC molecules. (0.05 MB DOC)

Reaction scheme of complex formation via a TNB-mixed disulfide (0.16 MB TIF)

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of t...

Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiotreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glyc...

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-t...

In Staphylococcus aureus thioredoxin (Trx) it has been shown that mutation of the conserved active site tryptophan residue (Trp28) has a large effect on the protein stability, on the pKa of the nucleophilic cysteine and on the redox potential. Since these effects can either be due to the partially unfolding of the Trp28Ala mutant or to the absence...

Nucleofugality is a measure of the quality of a leaving group in substitution and elimination reactions. In a conceptual DFT context, the nucleofugality is calculated for an elaborate set of common organic leaving groups, both in the gas phase and in two organic solvents (dichloromethane and methanol). An intrinsic nucleofugality scale is construct...

Conceptual DFT aims at describing the properties of molecules in interactions by using chemical reactivity descriptors. Herein, the redox behaviour of a given species, as quantified by the redox potential, is linked to DFT-based descriptors. We made use of a hierarchical decomposition of the corresponding half-reactions into one-electron reduction,...

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphyloc...

The P31T mutant of Staphylococcus aureus thioredoxin crystallizes spontaneously in space group P2(1)2(1)2(1), with unit-cell parameters a = 41.7, b = 49.5, c = 55.6 A. The crystals diffract to 2.2 A resolution. Isomorphous crystals of wild-type thioredoxin as well as of other point mutants only grow when seeded with the P31T mutant. These results s...

In the thioredoxin (Trx)-coupled arsenate reductase family, arsenate reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disu...

The reduction of arsenate to arsenite by pI258 arsenate reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of arsenate by pI258 A...

It is well documented that helices in proteins can decrease the pKa of residues located at the N-terminus, but the real nature of this perturbation remains unclear. In the present work, the origin of the effect of 3(10)- and alpha-polyalanine helices on the pKa of an N-terminal cysteine residue is examined in gas phase as well as in aqueous solutio...

Multiply charged anions (MCA's) are unstable relative to electron autoejection; however, the repulsive Coulomb barrier (RCB) provides electronic stability. In view of their interest in biological systems, the behavior of isolated AsO(4)(3-), PO(4)(3-), SO(4)(2-), and SeO(4)(2-) in the gas phase and in solution has been studied. To calculate the RCB...

The first step in the reduction of arsenate to arsenite catalyzed by the enzyme arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 involves the nucleophilic attack of a cysteine thiolate (Cys10) on the arsenic atom, leading to a covalent sulfur-arseno intermediate. We present a quantum chemical study on the onset of the nucleophilic...

and Vrije UniVersiteit Brussel (VUB), Dienst Ultrastructuur, Vlaams interuniVersitair Instituut Voor Biotechnologie (VIB), Pleinlaan 2, B-1050, Brussels, Belgium ReceiVed: February 13, 2003; In Final Form: May 15, 2003 We present a DFT study on the reactivity of an alkoxide and a thiolate toward the neutral and all ionic forms of arsenate and phosp...