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Dive into the research topics where Pierre Lafite is active.

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Featured researches published by Pierre Lafite.


Nature | 2011

The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase

Dea Slade; Mark S. Dunstan; Eva Barkauskaite; Ria Weston; Pierre Lafite; Neil Dixon; Marijan Ahel; David Leys; Ivan Ahel

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.


Journal of Biological Chemistry | 2009

The structure of Mycobacterium tuberculosis CYP125: Molecular basis for cholesterol binding in a P450 needed for host infection

Kirsty J. McLean; Pierre Lafite; Colin Levy; Myles R. Cheesman; Natalia Mast; Irina A. Pikuleva; David Leys; Andrew W. Munro

We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice. CYP125 is purified in a high spin form and undergoes both type I and II spectral shifts with various azole drugs. The 1.4-Å structure of ligand-free CYP125 reveals a “letterbox” active site cavity of dimensions appropriate for entry of a polycyclic sterol. A mixture of hexa-coordinate and penta-coordinate states could be discerned, with water binding as the 6th heme-ligand linked to conformation of the I-helix Val267 residue. Structures in complex with androstenedione and the antitubercular drug econazole reveal that binding of hydrophobic ligands occurs within the active site cavity. Due to the funnel shape of the active site near the heme, neither approaches the heme iron. A model of the cholesterol CYP125 complex shows that the alkyl side chain extends toward the heme iron, predicting hydroxylation of cholesterol C27. The alkyl chain is in close contact to Val267, suggesting a substrate binding-induced low- to high-spin transition coupled to reorientation of the latter residue. Reconstitution of CYP125 activity with a redox partner system revealed exclusively cholesterol 27-hydroxylation, consistent with structure and modeling. This activity may enable catabolism of host cholesterol or generation of immunomodulatory compounds that enable persistence in the host. This study reveals structural and catalytic properties of a potential M. tuberculosis drug target enzyme, and the likely mode by which the host-derived substrate is bound and hydroxylated.


Nature | 2013

Structural basis of kynurenine 3-monooxygenase inhibition.

Marta Amaral; Colin Levy; Derren J. Heyes; Pierre Lafite; Tiago F. Outeiro; Flaviano Giorgini; David Leys; Nigel S. Scrutton

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington’s-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer’s disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO–UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood–brain barrier in targeted therapies against neurodegenerative diseases such as Huntington’s, Alzheimer’s and Parkinson’s diseases.


Journal of Biological Chemistry | 2010

Structural and Biochemical Characterization of Mycobacterium tuberculosis CYP142 EVIDENCE FOR MULTIPLE CHOLESTEROL 27-HYDROXYLASE ACTIVITIES IN A HUMAN PATHOGEN

Max D. Driscoll; Kirsty J. McLean; Colin Levy; Natalia Mast; Irina A. Pikuleva; Pierre Lafite; Stephen E. J. Rigby; David Leys; Andrew W. Munro

The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å, revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ΔCYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism.


Nature Communications | 2013

Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Eva Barkauskaite; Amy Brassington; Edwin S. Tan; Jim Warwicker; Mark S. Dunstan; Benito Banos; Pierre Lafite; Marijan Ahel; Timothy J. Mitchison; Ivan Ahel; David Leys

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.


Analytica Chimica Acta | 2012

New development in in-capillary electrophoresis techniques for kinetic and inhibition study of enzymes.

Hala Nehmé; Reine Nehmé; Pierre Lafite; Sylvain Routier; Philippe Morin

Enzymes are often quantified by measuring their biological activity. Capillary electrophoresis is gaining its position in this field due to the ongoing trend to miniaturize biochemical assays. The aim of this work was to compare pre-capillary (off-line) and in-capillary electrophoresis techniques for studying enzymatic activity. The β-galactosidase (β-Gal) was chosen as a model enzyme. Each technique was optimized independently in order to decrease analyte consumption (to few tens of nanoliters), incubation time (to few seconds) and analysis time (below 1 min). Several experimental parameters (ionic strength of the background electrolyte (BGE) and of the incubation buffer, incubation time, injected volumes, …) were optimized by following peak efficiencies, resolution and repeatability. To monitor the performance of each technique, the catalytic constants (V(max) and K(m)) of 4-nitro-phenyl-d-galactopyranoside (PNPG) hydrolysis by β-Gal as well as the inhibition constants (K(i) and IC(50)) by a competitive inhibitor 2-nitrophenyl-1-thio-β-d-thiogalactopyranoside (ONPTG) were determined. The results obtained were cross compared and were also evaluated by comparison to a standard spectrophotometric method. EMMA proved to be the best technique in terms of sample consumption and speed. The short-end injection was successfully used which speeded-up electrophoretic analysis (<0.8 min). It is a very powerful tool for studying enzymatic inhibition. Usually, the inhibitor is injected in the capillary mixed to the substrate especially when both have similar mobilities. We show in this work, for the first time, that combining at-inlet reaction with EMMA-CE allows enzyme inhibition to be realized without any prior mixing of the substrate and the inhibitor. This approach is very interesting for screening inhibitors, rapidly and without excessive substrate consumption.


ChemBioChem | 2009

Parallel pathways and free-energy landscapes for enzymatic hydride transfer probed by hydrostatic pressure

Christopher R. Pudney; Tom McGrory; Pierre Lafite; Jiayun Pang; Sam Hay; David Leys; Michael J. Sutcliffe; Nigel S. Scrutton

Mutation of an active‐site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor–acceptor distance in the wild‐type enzyme, increased conformational freedom “dampens” its effect in the mutant.


Journal of Biological Chemistry | 2009

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase CONVERGENT EVOLUTION OF A SUGAR-ACTIVATING ENZYME WITH DNA/RNA POLYMERASES

Derren J. Heyes; Colin Levy; Pierre Lafite; Ian S. Roberts; Marie Goldrick; Andrew V. Stachulski; Steven R. Rossington; Deborah Stanford; Stephen E. J. Rigby; Nigel S. Scrutton; David Leys

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg2+ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp100 and Asp235 in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.


Journal of Biological Chemistry | 2009

An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

Tewes Tralau; Pierre Lafite; Colin Levy; John P. Combe; Nigel S. Scrutton; David Leys

We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehydrogenases. Using a novel synthetic in vivo reporter system for cellular formaldehyde, we found that the oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channeling mechanism. We also showed that uncoupling of the active sites could be achieved by mutagenesis or deletion of the 5,10-methylenetetrahydrofolate synthase site and that this leads to accumulation of intracellular formaldehyde. Channeling occurs by nonbiased diffusion of the labile intermediate through a large solvent cavity connecting both active sites. This central “reaction chamber” is created by a modular protein architecture that appears primitive when compared with the sophisticated design of other paradigm substrate-channeling enzymes. The evolutionary origins of the latter were likely similar to dimethylglycine oxidase. This work demonstrates the utility of synthetic biology approaches to the study of enzyme mechanisms in vivo and points to novel channeling mechanisms that protect the cell milieu from potentially toxic reaction products.


Biochemistry | 2014

Unraveling the Substrate Recognition Mechanism and Specificity of the Unusual Glycosyl Hydrolase Family 29 BT2192 from Bacteroides thetaiotaomicron

Laure Guillotin; Pierre Lafite; Richard Daniellou

Glycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining α-l-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak α-l-fucosidase and β-d-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-d-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate α-l-fucose from binding efficiently to the fucosidase-like active site relative to other GH29 enzymes. β-d-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewis(X) antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6-7 Å apart.

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David Leys

University of Manchester

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Daniel Mansuy

Paris Descartes University

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Colin Levy

University of Manchester

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Hala Nehmé

University of Orléans

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Darryl C. Zeldin

National Institutes of Health

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