Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes

Functionalized artificial bidomain proteins based on an α-solenoid protein repeat scaffold : a new class of artificial diels-alderases

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3' domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels-Alder (D-A) reaction. This allowed us to obtain four new artificial Diels-Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV-vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D-A reaction. They were found to be able to catalyze the enantioselective D-A reaction of azachalcone with cyclopentadiene with up to 38% yield and 52% enantiomeric excess, which validates the proposed strategy. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggest that artificial metalloenzymes based on bidomain A3-A3 proteins modified with nitrogen donor ligands may be suitable for further catalyst optimization and may constitute valuable tools toward more efficient and selective artificial biocatalysts

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Conception et élaboration de nouvelles métalloenzymes artificielles selon la stratégie du cheval de Troie

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Elaboration de nouvelles métalloenzymes artificielles à partir de métalloporphyrines et d'anticorps monoclonaux

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Modélisation du site actif de la superoxyde dismutase à manganèse (synthèse de ligands N-tripodaux, préparation de complexes de manganèse, caractérisations structurales et physico-chimiques)

Les superoxyde dismutases (SOD) protègent la cellule contre le superoxyde, en catalysant sa dismutation en dioxygène et peroxyde d'hydrogène. L'objectif de cette thèse consistait en la synthèse, la caractérisation et l'étude de la réactivité vis-à-vis du superoxyde de complexes de manganèse mimes de SOD. Les ligands choisis pour préparer ces complexes biomimétiques sont des ligands tripodaux à centre azoté, reproduisant le site actif des superoxyde dismutases à manganèse. Des complexes de manganèse ont été cristallisés et caractérisés par diffraction des rayons X, par spectroscopie UV-visible et RPE et par voltamétrie cyclique. Les structures obtenues sont variées et les propriétés magnétiques des complexes présentant des ponts carboxylato ont été étudiées. Dans le DMSO anhydre, les complexes [Mn(IPG)(CH3OH)]n(PF6)n et [Mn(BIG)(H20)2]2(PF6)2 réagissent avec le superoxyde en formant des dimères di-mu-oxo [MnIII(O)2MnIV]+, ce qui montre que le superoxyde se fixe au manganèse(II). Les complexes [Mn(BMPG)(H2O)]n(PF6)n et [Mn(TMIMA)2](PF6)2 réagissent avec le superoxyde sans former de dimères, probablement en raison de l'encombrement de leur sphère de coordination. La présence de molécules d'eau fixées au manganèse provoque un décalage dans la stœchiométrie des réactions observées. Les IC50 des complexes sont comprises entre 0,7 et 4,2.10^-6 mol.L^-1, prouvant que les complexes synthétisés sont de bons modèles de SOD en solution aqueuse. Une corrélation linéaire établie entre le potentiel anodique des complexes et l'activité montre que l'étape cinétiquement limitante est l'oxydation du manganèse(II). Enfin, par radiolyse pulsée, les constantes de vitesse k_cat ont été mesurées pour deux complexes et deux intermédiaires réactionnels, dont un adduit [MnII(IPG)O2] se formant à la vitesse de 4.10^8 L.mol^-1.s^-1, ont été mis en évidence lors de la réaction de [Mn(IPG)(CH3OH)]n(PF6)n avec le superoxyde. Un mécanisme catalytique détaillé a été proposé pour ce complexe.Superoxide dismutases (SOD) protect cells against superoxide as they catalyse its dismutation to oxygen and hydrogen peroxide. The purpose of this work was the synthesis of biomimetic manganese complexes, their characterization and the study of their reactivity towards superoxide. Nitrogen-centered tripodal ligands mimicking the active site of manganese SOD have been chosen. Manganese complexes have been crystallised and characterised by X-ray diffraction, electronic and EPR spectroscopy and by cyclic voltammetry. Obtained structures are various, and magnetic properties of complexes with carboxylato bridges have been studied. In anhydrous DMSO, the complexes [Mn(IPG)(CH3OH)]n(PF6)n and [Mn(BIG)(H2O)2]2(PF6)2 react with superoxide and form di-mu-oxo dimers [MnIII(O)2MnIV]+, showing that superoxide enters the coordination sphere of manganese(II). The complexes [Mn(BMPG)(H20)]n(PF6)n and [Mn(TMIMA)2](PF6)2 react with superoxide without the formation of any dimer, probably owing to the steric congestion of the metal environment. The presence of water linked to manganese induce a difference in the observed stoechiometry that have been rationalised. IC50 values range between 0.7 and 4.2.10^-6 mol.L^-1, demonstrating that synthesised complexes are good SOD mimics in aqueous solution. The linear correlation obtained between the anodic potential of complexes and their activity shows that the kinetically limiting step is the manganese(II) oxidation. Finally, by pulse radiolysis, kinetic constants k_cat for two complexes have been measured and two intermediates, one being an adduct [MnII(IPG)O2] formed with a rate of 4.10^8 L.mol^-1.s^-1, have been characterised during the reaction of [Mn(IPG)(CH3OH)]n(PF6)n with superoxide. A detailed catalytic mechanism have been proposed.TOULON-BU Centrale (830622101) / SudocORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Synthèse et évaluations enzymatiques de nouveaux inhibiteurs des ribose-5-phosphate isomérases de type A et B (étude cristallographique de complexes enzyme-inhibiteur chez Mycobacterium tuberculosis et implications mécanistiques)

LES RIBOSE-5-PHOSPHATE ISOMERASES (RPI A ET B) SONT DES ENZYMES CATALYSANT L'ISOMÉRISATION RÉVERSIBLE ENTRE LE RIBOSE 5-PHOSPHATE ET LE RIBULOSE 5-PHOSPHATE. ELLES SONT IMPLIQUÉES DANS DIFFÉRENTES VOIES MÉTABOLIQUES TELLES QUE LA VOIE DES PENTOSES PHOSPHATES OU LE CYCLE DE CALVIN... LA RÉACTION EST SUPPOSÉE IMPLIQUER UN INTERMÉDIAIRE DE HAUTE ÉNERGIE (IHE) DE TYPE 1,2-CIS-ÈNEDIOL(ATE). LA SYNTHÉSE DE NOUVEAUX INHIBITEURS COMPÉTITIFS DES RPI A ÉTÉ RÉALISÉE PAR DES STRATÉGIES DE PROTECTIONS / DÉPROTECTIONS FAISANT INTERVENIR LE D-ARABINOSE COMME PRODUIT DE DÉPART. LES PROPRIÉTÉS INHIBITRICES DES PRODUITS SYNTHÉTISÉS ONT ÉTÉ ÉVALUÉES SUR LA RPIA D'ÉPINARD, LA RPIB DE MYCOBACTERIUM TUBERCULOSIS ET LA RPIB DE TRYPANOSOMA CRUZI. L'ACIDE 4-DEOXY-4-PHOSPHO-D-ÉRYTHRONOHYDROXAMIQUE (4PEH) APPARÂIT COMME UN INHIBITEUR AUSSI PUISSANT QUE LE 4-DEOXY-4-PHOSPHO-D-ÉRYTHRONATE (4PEA) CHEZ LES RPIA ET COMME LE PLUS PUISSANT INHIBITEUR DE RPIB RAPPORTÉ A CE JOUR. LE 4-DEOXY-4-PHOSPHONOMETHYL-D-ÉRYTHRONATE (4PMEA) EST LE PREMIER ANALOGUE ISOSTÈRE STABLE DU 4PEA AYAN UN POUVOIR D'INHIBITION IMPORTANT SUR LES RPIA ET B. L'ENSEMBLE DES RÉSULTATS OBTENUS CONFORTENT L'IDÉE QUE LE MÉCANISME ENZYMATIQUE PROCÈDE VIA UN IHE DE TYPE 1,2-CIS-ÈNEDIOLATE. LES STRUCTURES 3D RÉSOLUES A 2.1 ET 2.2 A DE LA RPIB DE MYCOBACTERIUM TUBERCULOSIS COMPLEXÉE RESPECTIVEMENT AU 4PEH ET 4PEA SONT DECRITES. CELLES-CI PERMETTENT DE TIRER DES CONCLUSIONS SUR LES DÉTAILS DU MÉCANISME CATALYTIQUE: LE GLU75 SEMBLE ÊTRE LA BASE CATALYTIQUE IMPLIQUÉE DANS LE TRANSFERT DE PROTON ENTRE LES ATOMES DE CARBONE C1 ET C2, DE MEME QUE LA SER71 EST DISPOSÉE POUR ASSURER LE TRANSFERT DE PROTON ENTRE LES ATOMES D'OXYGÈNE O1 ET O2.RIBOSE-5-PHOSPHATE ISOMERASES (RPI A AND B) ARE ENZYMES CATALYSING THE REVERSIBLE CONVERSION BETWEEN RIBOSE 5-PHOSPHATE AND RIBULOSE 5-PHOSPHATE. THEY ARE INVOLVED IN DIFFERENT METABOLIC PATHWAYS SUCH AS PENTOSE PHOSPHATE WAY OR CALVIN CYCLE... THE SUPPOSED REACTION MECHANISM IS THOUGHT TO INVOLVED A HIGH ENERGY INTERMEDIATE (HEl) OF 1 ,2-CIS-ENEDIOL(ATE) TYPE. THE SYNTHESIS OF NEW COMPETITIVE INHIBITORS OF RPI HAVE BEEN REALISED USING PROTECTION/DEPROTECTION STRATEGIES STARTING FROM D-ARABINOSE. THE EFFECTS OF THESE COMPOUNDS ON SPINACH RPIA, IN COMPARISON WITH MYCOBACTERIUM TUBERCULOSIS AND TRYPANOSOMA CRUZI RPIB, WERE CARRIED OUT. 4-DEOXY-4-PHOSPHO-D-ÉRYTHRONOHYDROXAMIC ACID (4PEH) APPEARS TO BE AS POTENT AS 4-DEOXY-4-PHOSPHO-D-ÉRYTHRONIC ACID (4PEA) ON RPIA AND AS THE MOST POTENT INHIBITOR EVER EVALUATED ON RPIB. 4-DEOXY-4-PHOSPHONOMETHYL-D-ÉRYTHRONIC ACID (4PMEA) IS THE FIRST HYDROLYTICALLY STABLE AND POTENT INHIBITOR EVER DESCRIBED FOR RPI. THE RESULTS OBTAINED STRONGLY SUPPORT THE IDEA THAT THE ENZYME MECHANISM INVQLVES AN ANIONIC HEI: 1,2-CIS-ENEDIOLATE. THE 3-D STRUCTURES RESOLVED AT 2.1 AND 2.2 A OF RPIB FROM MYCOBACTERIUM TUBERCULOSIS COMPLEXED RESPECTIVELY WITH 4PEH ET 4PEA ARE DESCRIBED. THESE PERMIT TO DRAW CONCLUSIONS ABOUT DETAILS OF THE CATALYTIC MECHANISM : GLU75 SEEMS TO BE THE CATALYTIC BASE INVOLVED IN PROTON TRANSFERT BETWEEN CARBON ATOMS C1 AND C2, AND SER71 IS WELL LOCATED TO ENSURE PROTON TRANSFERT BETWEEN OXYGEN ATOMES O1 AND O2.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Elaboration d'une nouvelle génération d'hémoprotéines (couplage spécifique d'un cofacteur métalloporphyrinique sur un anticorps monoclonal)

Une nouvelle approche a été proposée afin de préparer une nouvelle génération d'hémoprotéines, catalyseurs d'oxydations sélectives. Elle consiste à modifier un anticorps (Ac) anti-substrat, un anti-œstradiol, en lui greffant covalemment une métalloporphyrine, pour obtenir un système capable d'oxyder sélectivement des stéroïdes. La principale difficulté de cette approche réside dans le contrôle de la fixation du cofacteur à proximité au site de liaison de l'Ac. Pour mener à bien cet objectif, une stratégie en 2 phases a été développée. La première phase a consisté à étudier un potentiel site de fixation du cofacteur à proximité du site de liaison de l'Ac. D'après la littérature, le choix s'est porté sur les lysines. Afin de répertorier les résidus potentiellement fonctionnalisables, un dosage préalable des lysines accessibles ainsi que des études structurales du paratope de l'Ac ont été réalisés. Après caractérisation de la structure primaire de ce domaine, il a été possible de réaliser une modélisation 3D de cette région permettant ainsi de visualiser les lysines les plus accessibles. La deuxième phase du projet a consisté à mettre au point une molécule permettant de coupler spécifiquement le cofacteur sur l'Ac, composée d'un motif antigénique, une testostérone, couplé à une métallo-méso-tétraarylporphyrine. Pour cela, une porphyrine disubstituée en ortho a été préparée afin de lier, d'un côté le stéroïde modifié et de l'autre, l'Ac anti-stéroïde au voisinage du site de reconnaissance de l'Ac. En parallèle, la testostérone a été modifiée dans le but de lui adjoindre un bras espaceur comportant un pont di sulfure qui se situera entre le stéroïde et le cofacteur. Ce pont disulfure utilisé comme fonction sécable permettra à la suite de sa réduction de relarguer la testostérone hors du site de reconnaissance de l'anticorps. L'étape finale de ce travail sera de coupler la métalloporphyrine à l'Ac afin d'étudier l'activité catalytique de ce nouveau biocatalyseur.We propose a strategy in order to prepare a new generation of hemoproteins as selective oxidation catalysts. This new approach involves the chemical labeling of antisubstrate antibodies (Ab) with a metalloporphyrin. The developed example consists in the covalent linkage of a metalloporphyrin to an antiestradiol Ab, in order to obtain semisynthetic catalytic Abs able to catalyze the selective oxidation of steroids. The main issue of this strategy is the control of the cofactor coupling in vicinity of the Ab- binding site. Therefore, we have developed a strategy which involves two phases. First, a potential coupling site had to be determined. According to the literature, we chose lysyl residues. In order to index the potentially derivatisable lysines, we undertook a quantification of available residues and preliminary structural studies about the Ab paratope. After characterisation of the primary structure of the Ab-binding site, we modelised this region allowing us to visualize the most convenient lysines for our strategy. The second phase involves the design and preparation of a molecule consisting of an antigenic steroid moiety, a testosterone, and a metallo-meso-tetra-arylporphyrin allowing to specifically couple the cofactor to the Ab. An ortho bisubstituted porphyrin has been prepared to link in one hand a modified testosterone and in the other hand, the Ab, nearby its binding site. In parallel, the testosterone moiety has been functionalized with a spacer group including a disulfide bridge. This bridge will be used as a cleavable linkage between the steroid and the cofactor in the final molecule, so that the coupled testosterone could be released from the conjugate after reduction of this function and be easily removed by dialysis. The final aim of this work will be to couple the metalloporphyrin to the Ab and to study the catalytic activity of this new biocatalyst.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Electronic and Steric Effects in Cobalt Schiff Bases Complexes: Synthesis, Characterization and Catalytic Activity of Some Cobalt(II) Tetra-Halogens-Dimethyl Salen Complexes

International audienceThe synthesis, characterization and catalytic activity of a series of tetra-halogeno-dimethyl salen cobalt (II) complexes are reported in this paper. The investigated complexes of cobalt (II) with Schiff bases are: αα'-di-methyl Salen cobalt (II) [Co(dMeSalen)], 3,3',5,5'-tetra chloro α,α'-di-methyl Salen cobalt (II), [Co(tCldMeSalen)], 3,3'-di-bromo 5,5'-di-chloro α,α'-di-methyl Salen cobalt (II), [Co(tBrdMeSalen)], 3,3',5,5'-tetra bromo α,α'-di-methyl Salen cobalt (II), [Co(tBrdMeSalen)] and 3,3',5,5'-tetra iodo α,α'-di-methyl Salen cobalt (II), [Co(tIdMeSalen)] (where Salen is bis(salicylaldehyde)ethylenediamine). The characterization of the complexes was performed by elemental analysis, cyclic voltammetry, UV-Vis, IR and EPR spectroscopies. The study was made in DMF, and pyridine was used for coordination as axial base. The redox potential is influenced by the substituent grafted on aromatic ring and in the azomethynic position and also by the molecules coordinating in axial position (solvent, DMF, or pyridine). The catalytic oxygenation of 2,6-di-tert-butylphenol by these complexes leads to the obtention of benzoquinone and diphenoquinone products. The cobalt (II) complexes form reversible adducts with molecular oxygen