I04-Macromolecular Crystallography
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Federico
Sabbadin
,
Saioa
Urresti
,
Bernard
Henrissat
,
Anna O.
Avrova
,
Lydia R. J.
Welsh
,
Peter J.
Lindley
,
Michael
Csukai
,
Julie N.
Squires
,
Paul H.
Walton
,
Gideon J.
Davies
,
Neil C.
Bruce
,
Stephen C.
Whisson
,
Simon J.
Mcqueen-Mason
Diamond Proposal Number(s):
[9948]
Abstract: The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans. We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.
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Aug 2021
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B21-High Throughput SAXS
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Ane
Rodrigo-Unzueta
,
Mattia
Ghirardello
,
Saioa
Urresti
,
Ignacio
Delso
,
David
Giganti
,
Itxaso
Anso
,
Beatriz
Trastoy
,
Natalia
Comino
,
Montse
Tersa
,
Cecilia
D'Angelo
,
Javier O.
Cifuente
,
Alberto
Marina
,
Jobst
Liebau
,
Lena
Mäler
,
Alexandre
Chenal
,
David
Albesa-Jove
,
Pedro
Merino
,
Marcelo E.
Guerin
Abstract: The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-β-strand and β-strand-to-α-helix transitions, and (ii) an ‘open-to-closed’ motion between the two Rossmann-fold domains, a conformational change necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such activation mechanism we analyzed a series of chemical derivatives, including mannose-phosphate (Man-P) and mannose-pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, as pyrophosphate-ribose (PP-RIB) and GMP, by the combined used of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry and Small Angle X-ray Scattering methods. Although the β-phosphate is present, we found that the mannose ring, neither covalently attached to phosphate (Man-P) nor to PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the ‘open-to-closed’ motion, with the β-phosphate group providing the high affinity binding to PimA. Altogether, the experimental data, contribute to a better understanding of the structural determinants involved in the ‘open-to-closed’ motion observed not only in PimA, but also visualized/predicted in other glycosyltransferases. In addition, the experimental data might prove useful for the discovery/development of PimA and/or glycosyltransferase inhibitors.
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Jul 2020
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The recent discovery of `lytic' polysaccharide monooxygenases, copper-dependent enzymes for biomass degradation, has provided new impetus for the analysis of unusual metal-ion sites in carbohydrate-active enzymes. In this context, the CAZY family GH124 endoglucanase from Ruminiclostridium thermocellum contains an unusual metal-ion site, which was originally modelled as a Ca2+ site but features aspartic acid, asparagine and two histidine imidazoles as coordinating residues, which are more consistent with a transition-metal binding environment. It was sought to analyse whether the GH124 metal-ion site might accommodate other metals. It is demonstrated through thermal unfolding experiments that this metal-ion site can accommodate a range of transition metals (Fe2+, Cu2+, Mn2+ and Ni2+), whilst the three-dimensional structure and mass spectrometry show that one of the histidines is partially covalently modified and is present as a 2-oxohistidine residue; a feature that is rarely observed but that is believed to be involved in an `off-switch' to transition-metal binding. Atomic resolution (<1.1 Å) complexes define the metal-ion site and also reveal the binding of an unusual fructosylated oligosaccharide, which was presumably present as a contaminant in the cellohexaose used for crystallization. Although it has not been possible to detect a biological role for the unusual metal-ion site, this work highlights the need to study some of the many metal-ion sites in carbohydrate-active enzymes that have long been overlooked or previously mis-assigned.
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Aug 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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David
Albesa-Jove
,
Javier
Romero-Garcia
,
Enea
Sancho-Vaello
,
F.-Xabier
Contreras
,
Ane
Rodrigo-Unzueta
,
Natalia
Comino
,
Ana
Carreras-Gonzalez
,
Pedro
Arrasate
,
Saioa
Urresti
,
Xevi
Biarnes
,
Antoni
Planas
,
Marcelo
Guerin
Diamond Proposal Number(s):
[8302, 10130]
Abstract: Glycosyltransferases (GTs) play a central role in nature. They catalyze the transfer of a sugar moiety to a broad range of acceptor substrates. GTs are highly selective enzymes, allowing the recognition of subtle structural differences in the sequences and stereochemistry of their sugar and acceptor substrates. We report here a series of structural snapshots of the reaction center of the retaining glucosyl-3-phosphoglycerate synthase (GpgS). During this sequence of events, we visualize how the enzyme guides the substrates into the reaction center where the glycosyl transfer reaction takes place, and unveil the mechanism of product release, involving multiple conformational changes not only in the substrates/products but also in the enzyme. The structural data are further complemented by metadynamics free-energy calculations, revealing how the equilibrium of loop conformations is modulated along these itineraries. The information reported here represent an important contribution for the understanding of GT enzymes at the molecular level.
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Jun 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Delu (tyler)
Yin
,
Saioa
Urresti
,
Mickael
Lafond
,
Esther M.
Johnston
,
Fatemeh
Derikvand
,
Luisa
Ciano
,
Jean- Guy
Berrin
,
Bernard
Henrissat
,
Paul
Walton
,
Gideon
Davies
,
Harry
Brumer
Diamond Proposal Number(s):
[9948]
Abstract: Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure-function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-a-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications.
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Dec 2015
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I04-Macromolecular Crystallography
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David
Albesa-Jove
,
Natalia
Comino
,
Montse
Tersa
,
Elisabeth
Mohorko
,
Saioa
Urresti
,
Elisa
Dainese
,
Laurent R.
Chiarelli
,
Maria Rosalia
Pasca
,
Riccardo
Manganelli
,
Vadim
Makarov
,
Giovanna
Riccardi
,
Dmitri I.
Svergun
,
Rudi
Glockshuber
,
Marcelo
Guerin
Diamond Proposal Number(s):
[8302, 10130]
Open Access
Abstract: Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a ′closed′ compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an ′open′ conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.
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Nov 2015
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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David
Albesa-Jové
,
Fernanda
Mendoza
,
Ane
Rodrigo-Unzueta
,
Fernando
Gomollón-Bel
,
Javier O.
Cifuente
,
Saioa
Urresti
,
Natalia
Comino
,
Hansel
Gómez
,
Javier
Romero-García
,
José M.
Lluch
,
Enea
Sancho-Vaello
,
Xevi
Biarnés
,
Antoni
Planas
,
Pedro
Merino
,
Laura
Masgrau
,
Marcelo
Guerin
Diamond Proposal Number(s):
[8302, 10130]
Abstract: Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host-pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. A native ternary complex of a GT in a productive mode for catalysis is reported, that of the retaining glucosyl-3-phosphoglycerate synthase GpgS from M. tuberculosis in the presence of the sugar donor UDP-Glc, the acceptor substrate phosphoglycerate, and the divalent cation cofactor. Through a combination of structural, chemical, enzymatic, molecular dynamics, and quantum-mechanics/molecular-mechanics (QM/MM) calculations, the catalytic mechanism was unraveled, thereby providing a strong experimental support for a front-side substrate-assisted SN i-type reaction.
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Aug 2015
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I04-Macromolecular Crystallography
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David
Giganti
,
David
Albesa-Jove
,
Saioa
Urresti
,
Ane
Rodrigo-Unzueta
,
Mariano A
Martínez
,
Natalia
Comino
,
Nathalie
Barilone
,
Marco
Bellinzoni
,
Alexandre
Chenal
,
Marcelo
Guerin
,
Pedro M.
Alzari
Diamond Proposal Number(s):
[8302, 10130]
Abstract: Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including β-strand–to–α-helix and α-helix–to–β-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.
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Nov 2014
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I02-Macromolecular Crystallography
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David
Albesa-Jove
,
Laurent R.
Chiarelli
,
Vadim
Makarov
,
Maria Rosalia
Pasca
,
Saioa
Urresti
,
Giorgia
Mori
,
Elena
Salina
,
Anthony
Vocat
,
Natalia
Comino
,
Elisabeth
Mohorko
,
Svetlana
Ryabova
,
Bernhard
Pfieiffer
,
Ana Luisa De Jesus
Lopes Ribeiro
,
Ane
Rodrigo-Unzueta
,
Montse
Tersa
,
Giuseppe
Zanoni
,
Silvia
Buroni
,
Karl-Heinz
Altmann
,
Ruben C.
Hartkoorn
,
Rudi
Glockshuber
Diamond Proposal Number(s):
[8302]
Abstract: The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 angstrom resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a beta-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti- mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.
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Jul 2014
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I04-Macromolecular Crystallography
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S.
Urresti
,
D.
Albesa-Jove
,
F.
Schaeffer
,
H. T.
Pham
,
D.
Kaur
,
P.
Gest
,
M. J.
Van Der Woerd
,
A.
Carreras-Gonzalez
,
S.
Lopez-Fernandez
,
P. M.
Alzari
,
P. J.
Brennan
,
M.
Jackson
,
M. E.
Guerin
Diamond Proposal Number(s):
[8302]
Open Access
Abstract: Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.
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Jul 2012
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