Publications by authors named "Carme Rovira"

104 Publications

Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-l-Arabinofuranosidase Inhibitor.

Angew Chem Int Ed Engl 2021 03 2;60(11):5754-5758. Epub 2021 Feb 2.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York, YO10 5DD, UK.

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys) (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived β-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This β-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
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http://dx.doi.org/10.1002/anie.202013920DOI Listing
March 2021

Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations.

Nat Commun 2021 01 14;12(1):367. Epub 2021 Jan 14.

Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, 13083-100, Brazil.

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.
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http://dx.doi.org/10.1038/s41467-020-20620-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809346PMC
January 2021

How Oxygen Binding Enhances Long-Range Electron Transfer: Lessons From Reduction of Lytic Polysaccharide Monooxygenases by Cellobiose Dehydrogenase.

Angew Chem Int Ed Engl 2021 02 30;60(5):2385-2392. Epub 2020 Nov 30.

State Key Laboratory of Structural Chemistry of Solid Surface and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.

Long-range electron transfer (ET) in metalloenzymes is a general and fundamental process governing O activation and reduction. Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the oxidative cleavage of insoluble polysaccharides, but their reduction mechanism by cellobiose dehydrogenase (CDH), one of the most commonly used enzymatic electron donors, via long-range ET is still an enigma. Using multiscale simulations, we reveal that interprotein ET between CDH and LPMO is mediated by the heme propionates of CDH and solvent waters. We also show that oxygen binding to the copper center of LPMO is coupled with the long-range interprotein ET. This process, which is spin-regulated and enhanced by the presence of O , directly leads to LPMO-Cu -O , bypassing the formation of the generally assumed LPMO-Cu species. The uncovered ET mechanism rationalizes experimental observations and might have far-reaching implications for LPMO catalysis as well as the O - or CO-binding-enhanced long-range ET processes in other metalloenzymes.
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http://dx.doi.org/10.1002/anie.202011408DOI Listing
February 2021

Photocontrol of Endogenous Glycine Receptors In Vivo.

Cell Chem Biol 2020 11 25;27(11):1425-1433.e7. Epub 2020 Aug 25.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain; Catalan Institution for Research and Advanced Studies (ICREA), Barcelona 08003 Spain; CIBER-BBN, Madrid 28001 Spain. Electronic address:

Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.
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http://dx.doi.org/10.1016/j.chembiol.2020.08.005DOI Listing
November 2020

An Epoxide Intermediate in Glycosidase Catalysis.

ACS Cent Sci 2020 May 16;6(5):760-770. Epub 2020 Apr 16.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 -α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope () conformation. Kinetic isotope effects (/) for anomeric-H and anomeric-C support an oxocarbenium ion-like transition state, and that for C2-O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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http://dx.doi.org/10.1021/acscentsci.0c00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256955PMC
May 2020

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

J Am Chem Soc 2020 03 26;142(10):4648-4662. Epub 2020 Feb 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5DD, U.K.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.
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http://dx.doi.org/10.1021/jacs.9b11351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068720PMC
March 2020

Molecular Mechanisms of Oxygen Activation and Hydrogen Peroxide Formation in Lytic Polysaccharide Monooxygenases.

ACS Catal 2019 Jun 22;9(6):4958-4969. Epub 2019 Apr 22.

Departament de Química Inorgànica i Orgànica & IQTCUB, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes for the degradation of recalcitrant polysaccharides such as chitin and cellulose. Unlike classical hydrolytic enzymes (cellulases), LPMOs catalyze the cleavage of the glycosidic bond via an oxidative mechanism using oxygen and a reductant. The full enzymatic molecular mechanisms, starting from the initial electron transfer from a reductant to oxygen activation and hydrogen peroxide formation, are not yet understood. Using quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, we have uncovered the oxygen activation mechanisms by LPMO in the presence of ascorbic acid, one of the most-used reductants in LPMOs assays. Our simulations capture the sequential formation of Cu(II)-O and Cu(II)-OOH intermediates via facile H atom abstraction from ascorbate. By investigating all the possible reaction pathways from the Cu(II)-OOH intermediate, we ruled out Cu(II)-O formation via direct O-O cleavage of Cu(II)-OOH. Meanwhile, we identified a possible pathway in which the proximal O atom of Cu(II)-OOH abstracts a hydrogen atom from ascorbate, leading to Cu(I) and HO. The in-situ-generated HO either converts to LPMO-Cu(II)-O via a homolytic reaction, or diffuses into the bulk water in an uncoupled pathway. The competition of these two pathways is strongly dependent on the binding of the carbohydrate substrate, which plays a role in barricading the in-situ-generated HO molecule, preventing its diffusion from the active site into the bulk water. Based on the present results, we propose a catalytic cycle of LPMOs that is consistent with the experimental information available. In particular, it explains the enigmatic substrate dependence of the reactivity of the LPMO with HO.
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http://dx.doi.org/10.1021/acscatal.9b00778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007194PMC
June 2019

Structural and kinetic features of aldehyde dehydrogenase 1A (ALDH1A) subfamily members, cancer stem cell markers active in retinoic acid biosynthesis.

Arch Biochem Biophys 2020 03 7;681:108256. Epub 2020 Jan 7.

Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193, Bellaterra, Barcelona, Spain. Electronic address:

Aldehyde dehydrogenases catalyze the NAD(P)-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower K values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of k/K. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.
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http://dx.doi.org/10.1016/j.abb.2020.108256DOI Listing
March 2020

A Single Point Mutation Converts GH84 -GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence.

J Am Chem Soc 2020 02 24;142(5):2120-2124. Epub 2020 Jan 24.

Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) and Institut de Quı́mica Teòrica i Computacional (IQTCUB) , Universitat de Barcelona , Martí i Franquès 1 , 08028 Barcelona , Spain.

Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.
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http://dx.doi.org/10.1021/jacs.9b09655DOI Listing
February 2020

Mannosidase mechanism: at the intersection of conformation and catalysis.

Curr Opin Struct Biol 2020 06 28;62:79-92. Epub 2019 Dec 28.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Mannosidases are a diverse group of enzymes that are important in the biological processing of mannose-containing polysaccharides and complex glycoconjugates. They are found in 12 of the >160 sequence-based glycosidase families. We discuss evidence that nature has evolved a small set of common mechanisms that unite almost all of these mannosidase families. Broadly, mannosidases (and the closely related rhamnosidases) perform catalysis through just two conformations of the oxocarbenium ion-like transition state: a B (or enantiomeric B) boat and a H half-chair. This extends to a new family (GT108) of GDPMan-dependent β-1,2-mannosyltransferases/phosphorylases that perform mannosyl transfer through a boat conformation as well as some mannosidases that are metalloenzymes and require divalent cations for catalysis. Yet, among this commonality lies diversity. New evidence shows that one unique family (GH99) of mannosidases use an unusual mechanism involving anchimeric assistance via a 1,2-anhydro sugar (epoxide) intermediate.
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http://dx.doi.org/10.1016/j.sbi.2019.11.008DOI Listing
June 2020

Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.

Nat Catal 2019 Nov;2(12):1115-1123

Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.

D-Apiose is a -branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from , molecular dynamics simulations expanded by QM/MM calculations, and mutational-mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide coenzyme with retro-aldol/aldol chemistry for the sugar ring contraction. They further demonstrate that decarboxylation occurs only after the sugar ring opening and identify the thiol group of Cys100 in steering the sugar skeleton rearrangement by proton transfer to and from the C3'. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis.
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http://dx.doi.org/10.1038/s41929-019-0382-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914363PMC
November 2019

Modeling catalytic reaction mechanisms in glycoside hydrolases.

Curr Opin Chem Biol 2019 12 12;53:183-191. Epub 2019 Nov 12.

Departament de Química Inorgànica I Orgànica (secció de Química Orgànica) and Institut de Química Teòrica I Computacional (IQTCUB), Universitat de Barcelona, Martí I Franquès 1 08028 Barcelona, Spain; Institució Catalana de Recerca I Estudis Avançats (ICREA) Passeig Lluís Companys 23, 08010 Barcelona, Spain. Electronic address:

Modeling catalysis in carbohydrate-active enzymes is a daunting challenge because of the high flexibility and diversity of both enzymes and carbohydrates. Glycoside hydrolases (GHs) are an illustrative example, where conformational changes and subtle interactions have been shown to be critical for catalysis. GHs have pivotal roles in industry (e.g. biofuel or detergent production) and biomedicine (e.g. targets for cancer and diabetes), and thus, a huge effort is devoted to unveil their molecular mechanisms. Besides experimental techniques, computational methods have served to provide an in-depth understanding of GH mechanisms, capturing complex reaction coordinates and the conformational itineraries that substrates follow during the whole catalytic pathway, providing a framework that ultimately may assist the engineering of these enzymes and the design of new inhibitors.
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http://dx.doi.org/10.1016/j.cbpa.2019.09.007DOI Listing
December 2019

Electrochemically Gated Long-Distance Charge Transport in Photosystem I.

Angew Chem Int Ed Engl 2019 09 5;58(38):13280-13284. Epub 2019 Aug 5.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028, Barcelona, Spain.

The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic reactions that are coupled through redox mediators, including proteins and small molecules. The use of native and synthetic redox probes is key to understanding charge transport mechanisms and to the design of bioelectronic sensors and solar energy conversion devices. However, redox probes have limited tunability to exchange charge at the desired electrochemical potentials (energy levels) and at different protein sites. Herein, we take advantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi level and nanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI) complexes. Current-distance measurements at different potentiostatic conditions indicate that PSI supports long-distance transport that is electrochemically gated near the redox potential of P700, with current extending farther under hole injection conditions.
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http://dx.doi.org/10.1002/anie.201904374DOI Listing
September 2019

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Secretomes Using Activity-Based Probes.

ACS Cent Sci 2019 Jun 24;5(6):1067-1078. Epub 2019 May 24.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and -β-1,4-xylanases in the secretomes of , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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http://dx.doi.org/10.1021/acscentsci.9b00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598175PMC
June 2019

Fenton-Derived OH Radicals Enable the MPnS Enzyme to Convert 2-Hydroxyethylphosphonate to Methylphosphonate: Insights from Ab Initio QM/MM MD Simulations.

J Am Chem Soc 2019 06 4;141(23):9284-9291. Epub 2019 Jun 4.

Institute of Chemistry , The Hebrew University of Jerusalem , 9190407 Jerusalem , Israel.

The mechanism for dioxygen activation represents one of the core issues in metalloenzymes. In most cases, the activation of the O molecule requires additional electrons from an external reducant. However, nonheme hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS) are exceptional C-H oxygenases. Both enzymes do not utilize reductants, rather they employ directly iron(III)-superoxide species to initiate H-abstraction reactions and lead thereby to catalysis of the C-C cleavage in 2-hydroxyethylphosphonate (2-HEP). Using the recently characterized MPnS structure and QM(B3LYP)/MM-based metadynamics simulations, we deciphered the chemical mechanism for MPnS. Our simulations demonstrate O activation in MPnS is mediated by an adjacent Lysine residue (Lys28) in the active site, leading to an unusual H O intermediate in the reductant-independent nonheme MPnS enzyme. Furthermore, the so-generated H O intermediate is subsequently employed in a Fenton-type reaction, leading to a locked •OH radical that spontaneously attaches to the substrate carbonyl group. Meanwhile, the proton from the Fe(III)-OH is shuttled back to the deprotonated Lys28, affording the Fe(IV)-oxo species that is identified by experiment in HEPD. Thus, our calculations demonstrate an unusual proton-shuttle mechanism for O activation in metalloenzymes.
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http://dx.doi.org/10.1021/jacs.9b02659DOI Listing
June 2019

Conformational Itinerary of Sucrose During Hydrolysis by Retaining Amylosucrase.

Front Chem 2019 30;7:269. Epub 2019 Apr 30.

Departament de Quimica Inorgànica i Orgànica (Secció de Química Orgànica) and Institut de Quimica Teòrica i Computacional, Universitat de Barcelona, Martí i Franquès 1, Barcelona, Spain.

By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of amylosucrase (AS), a member of GH13 family. Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted conformation, as inferred from structural studies, or a distorted -like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs). We show that, even though the first step of the reaction (glycosylation) results in a conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step (deglycosylation), in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon. The two conformations ( ad ) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.
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http://dx.doi.org/10.3389/fchem.2019.00269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502901PMC
April 2019

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site.

Nat Commun 2019 05 20;10(1):2222. Epub 2019 May 20.

School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond South Australia, 5064, Australia.

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl β-D-glucoside and methyl 6-thio-β-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-β-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.
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http://dx.doi.org/10.1038/s41467-019-09691-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527550PMC
May 2019

A photoswitchable GABA receptor channel blocker.

Br J Pharmacol 2019 08 26;176(15):2661-2677. Epub 2019 Jun 26.

INSERM, INS, Institut de Neurosciences des Systèmes, Aix-Marseille University, Marseille, France.

Background And Purpose: Anion-selective Cys-loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride-selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light-controllable modulator of GABA receptors, we constructed azobenzene-nitrazepam (Azo-NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group.

Experimental Approach: The experiments were carried out on cultured cells expressing Cys-loop receptors of known subunit composition and in brain slices using patch-clamp. Site-directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo-NZ1.

Key Results: At visible light, being in trans-configuration, Azo-NZ1 blocked heteromeric α1/β2/γ2 GABA receptors, ρ2 GABA (GABA ), and α2 glycine receptors, whereas switching the compound into cis-state by UV illumination restored the activity. Azo-NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans-configuration, Azo-NZ1 blocks the Cl-selective ion pore of GABA receptors interacting mainly with the 2' level of the TM2 region.

Conclusions And Implications: Azo-NZ1 is a soluble light-driven Cl-channel blocker, which allows photo-modulation of the activity induced by anion-selective Cys-loop receptors. Azo-NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination.
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http://dx.doi.org/10.1111/bph.14689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609548PMC
August 2019

Glucose transport via the pseudomonad porin OprB: implications for the design of Trojan Horse anti-infectives.

Phys Chem Chem Phys 2019 Apr;21(16):8457-8463

Departament de Química Inorgànica i Orgànica and Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain.

Deciphering the transport through outer-membrane porins is crucial to understand how anti-infectives enter Gram-negative bacteria and perform their function. Here we elucidated the transport mechanism of substrates through the Pseudomonads sugar-specific porin OprB by means of multiscale modeling. We used molecular dynamics simulations to quantify the energetics of transport and thus a diffusion model to quantify the macroscopic flux of molecules through OprB. Our results show that Trp171 and several glutamate residues in the constriction region are key for the transport of glucose, the preferred natural substrate, through OprB. The unveiled transport mechanism suggests that 2-acetamido-1,2-dideoxynojirimycin (DNJ-NAc), an anti-infective structurally similar to glucose, can enter the cell via OprB. We quantified its energetics and macroscopic flux through OprB providing a comparative analysis with the natural substrate. Thus this pore can be considered as a promising gateway for exploiting the Trojan Horse strategy in pathogenic bacteria.
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http://dx.doi.org/10.1039/c9cp00778dDOI Listing
April 2019

Long distance electron transfer through the aqueous solution between redox partner proteins.

Nat Commun 2018 12 4;9(1):5157. Epub 2018 Dec 4.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Barcelona, 08028, Spain.

Despite the importance of electron transfer between redox proteins in photosynthesis and respiration, the inter-protein electron transfer rate between redox partner proteins has never been measured as a function of their separation in aqueous solution. Here, we use electrochemical tunneling spectroscopy to show that the current between two protein partners decays along more than 10 nm in the solution. Molecular dynamics simulations reveal a reduced ionic density and extended electric field in the volume confined between the proteins. The distance-decay factor and the calculated local barrier for electron transfer are regulated by the electrochemical potential applied to the proteins. Redox partners could use electrochemically gated, long distance electron transfer through the solution in order to conciliate high specificity with weak binding, thus keeping high turnover rates in the crowded environment of cells.
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http://dx.doi.org/10.1038/s41467-018-07499-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279779PMC
December 2018

Palladium-mediated enzyme activation suggests multiphase initiation of glycogenesis.

Nature 2018 11 24;563(7730):235-240. Epub 2018 Oct 24.

Department of Chemistry, University of Oxford, Oxford, UK.

Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease, and mouse knockout and inherited human mutations of GYG impair glycogen synthesis. GYG acts as a 'seed core' for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation 'shunt' process using on-protein C-C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG's use of sugar substrates. This reveals a tolerant but 'proof-read' mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.
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http://dx.doi.org/10.1038/s41586-018-0644-7DOI Listing
November 2018

Molecular-Scale Ligand Effects in Small Gold-Thiolate Nanoclusters.

J Am Chem Soc 2018 11 30;140(45):15430-15436. Epub 2018 Oct 30.

Department of Chemistry , Dalhousie University , Halifax , Nova Scotia B3H 4R2 , Canada.

Because of the small size and large surface area of thiolate-protected Au nanoclusters (NCs), the protecting ligands are expected to play a substantial role in modulating the structure and properties, particularly in the solution phase. However, little is known on how thiolate ligands explicitly modulate the structural properties of the NCs at atomic level, even though this information is critical for predicting the performance of Au NCs in application settings including as a catalyst interacting with small molecules and as a sensor interacting with biomolecular systems. Here, we report a combined experimental and theoretical study, using synchrotron X-ray spectroscopy and quantum mechanics/molecular mechanics simulations, that investigates how the protecting ligands impact the structure and properties of small Au(SR) NCs. Two representative ligand types, smaller aliphatic cyclohexanethiolate and larger hydrophilic glutathione, are selected, and their structures are followed experimentally in both solid and solution phases. It was found that cyclohexanethiolate ligands are significantly perturbed by toluene solvent molecules, resulting in structural changes that cause disorder on the surface of Au(SR) NCs. In particular, large surface cavities in the ligand shell are created by interactions between toluene and cyclohexanethiolate. The appearance of these small molecule-accessible sites on the  NC surface demonstrates the ability of Au NCs to act as a catalyst for organic phase reactions. In contrast, glutathione ligands encapsulate the Au NC core via intermolecular interactions, minimizing structural changes caused by interactions with water molecules. The much better protection from glutathione ligands imparts a rigidified surface and ligand structure, making the NCs desirable for biomedical applications due to the high stability and also offering a structural-based explanation for the enhanced photoluminescence often reported for glutathione-protected Au NCs.
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http://dx.doi.org/10.1021/jacs.8b09440DOI Listing
November 2018

Oxazoline or Oxazolinium Ion? The Protonation State and Conformation of the Reaction Intermediate of Chitinase Enzymes Revisited.

Chemistry 2018 Dec 29;24(72):19258-19265. Epub 2018 Nov 29.

Departament de Química Inorgànica i Orgànica, (secció de Química Orgànica) and Institut de Química Teòrica i, Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028, Barcelona, Spain.

The enzymatic hydrolysis of chitin, one of the most abundant carbohydrates in nature, is achieved by chitinases, enzymes of increasing importance in biomedicine and industry. Unlike most retaining glycosidases, family GH18 chitinases follow a substrate-assisted mechanism in which the 2-acetamido group of one N-acetylglucosamine monomer, rather than a basic residue of the enzyme, reacts with the sugar anomeric carbon, forming an intermediate that has been described as an oxazolinium ion. Based on QM/MM metadynamics simulations on chitinase B from Serratia marcescens, we show that the reaction intermediate of GH18 chitinases features instead a neutral oxazoline in a C / H conformation, with an oxazolinium ion being formed on the pathway towards the reaction products. The role of a well-defined hydrogen-bond network that operates around the N-acetyl group, orchestrating catalysis by protonation events, is discussed.
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http://dx.doi.org/10.1002/chem.201803905DOI Listing
December 2018

Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.

ACS Cent Sci 2018 Sep 14;4(9):1274-1290. Epub 2018 Sep 14.

BIFI, University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza 50018, Spain.

Mucin-type -glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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http://dx.doi.org/10.1021/acscentsci.8b00488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161044PMC
September 2018

Theory Uncovers the Role of the Methionine-Tyrosine-Tryptophan Radical Adduct in the Catalase Reaction of KatGs: O  Release Mediated by Proton-Coupled Electron Transfer.

Chemistry 2018 Apr 6;24(20):5388-5395. Epub 2018 Apr 6.

Departament de Química Inorgànica i Orgànica, (secció de Química Orgànica) &, Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028, Barcelona, Spain.

Catalase-peroxidases (KatGs) are bifunctional enzymes exhibiting both peroxidase and substantial catalase activities. It is widely recognized from experiments that the catalatic activity of KatGs is correlated with a unique covalent adduct (M-Y-W) formed in the active site, but the exact role of this adduct was elusive up to now. Here, quantum mechanical/molecular mechanical (QM/MM) calculations and QM/MM metadynamics are employed to elucidate the molecular mechanism and the role of M-Y-W adduct in the catalase reaction. It is shown that O formation proceeds through a mechanism involving proton-coupled electron transfer (PCET). The M-Y-W cation radical adduct, which is close to the heme, His112 and the HOO radical intermediate, acts as an electron sink during the PCET process. The present study also highlights the structural differences and functional similarities between KatGs and monofunctional catalases.
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http://dx.doi.org/10.1002/chem.201706076DOI Listing
April 2018

Redesigning the Coumarin Scaffold into Small Bright Fluorophores with Far-Red to Near-Infrared Emission and Large Stokes Shifts Useful for Cell Imaging.

J Org Chem 2018 02 11;83(3):1185-1195. Epub 2018 Jan 11.

Secció de Química Orgànica, Departament de Química Inorgànica i Orgànica, Universitat de Barcelona , Martí i Franquès 1-11, E-08028 Barcelona, Spain.

Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 μM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.
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http://dx.doi.org/10.1021/acs.joc.7b02660DOI Listing
February 2018

Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

ACS Cent Sci 2017 Nov 13;3(11):1168-1173. Epub 2017 Nov 13.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, His) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-His. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from and pantothenate synthetase.
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http://dx.doi.org/10.1021/acscentsci.7b00341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704290PMC
November 2017

The Molecular Mechanism of Substrate Recognition and Catalysis of the Membrane Acyltransferase PatA from Mycobacteria.

ACS Chem Biol 2018 01 11;13(1):131-140. Epub 2017 Dec 11.

Structural Biology Unit, CIC bioGUNE , Bizkaia Technology Park, 48160 Derio, Spain.

Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements, and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM/PIM. We report here the crystal structure of PatA in the presence of 6-O-palmitoyl-α-d-mannopyranoside, unraveling the acceptor binding mechanism. The acceptor mannose ring localizes in a cavity at the end of a surface-exposed long groove where the active site is located, whereas the palmitate moiety accommodates into a hydrophobic pocket deeply buried in the α/β core of the protein. Both fatty acyl chains of the PIM acceptor are essential for the reaction to take place, highlighting their critical role in the generation of a competent active site. By the use of combined structural and quantum-mechanics/molecular-mechanics (QM/MM) metadynamics, we unravel the catalytic mechanism of PatA at the atomic-electronic level. Our study provides a detailed structural rationale for a stepwise reaction, with the generation of a tetrahedral transition state for the rate-determining step. Finally, the crystal structure of PatA in the presence of β-d-mannopyranose and palmitate suggests an inhibitory mechanism for the enzyme, providing exciting possibilities for inhibitor design and the discovery of chemotherapeutic agents against this major human pathogen.
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http://dx.doi.org/10.1021/acschembio.7b00578DOI Listing
January 2018

1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor.

ACS Cent Sci 2017 Jul 13;3(7):784-793. Epub 2017 Jul 13.

Department of Bio-organic Synthesis and Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6--cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose C chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
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http://dx.doi.org/10.1021/acscentsci.7b00214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532717PMC
July 2017

An atypical interaction explains the high-affinity of a non-hydrolyzable S-linked 1,6-α-mannanase inhibitor.

Chem Commun (Camb) 2017 Aug 2;53(66):9238-9241. Epub 2017 Aug 2.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.

The non-hydrolyzable S-linked azasugars, 1,6-α-mannosylthio- and 1,6-α-mannobiosylthioisofagomine, were synthesized and shown to bind with high affinity to a family 76 endo-1,6-α-mannanase from Bacillus circulans. X-ray crystallography showed an atypical interaction of the isofagomine nitrogen with the catalytic acid/base. Molecular dynamics simulations reveal that the atypical binding results from sulfur perturbing the most stable form away from the nucleophile interaction preferred for the O-linked congener.
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http://dx.doi.org/10.1039/c7cc04977cDOI Listing
August 2017