RESUMO
Glycoside hydrolases (glycosidases) take part in myriad biological processes and are important therapeutic targets. Competitive and mechanism-based inhibitors are useful tools to dissect their biological role and comprise a good starting point for drug discovery. The natural product, cyclophellitol, a mechanism-based, covalent and irreversible retaining ß-glucosidase inhibitor has inspired the design of diverse α- and ß-glycosidase inhibitor and activity-based probe scaffolds. Here, we sought to deepen our understanding of the structural and functional requirements of cyclophellitol-type compounds for effective human α-glucosidase inhibition. We synthesized a comprehensive set of α-configured 1,2- and 1,5a-cyclophellitol analogues bearing a variety of electrophilic traps. The inhibitory potency of these compounds was assessed towards both lysosomal and ER retaining α-glucosidases. These studies revealed the 1,5a-cyclophellitols to be the most potent retaining α-glucosidase inhibitors, with the nature of the electrophile determining inhibitory mode of action (covalent or non-covalent). DFT calculations support the ability of the 1,5a-cyclophellitols, but not the 1,2-congeners, to adopt conformations that mimic either the Michaelis complex or transition state of α-glucosidases.
Assuntos
Inibidores de Glicosídeo Hidrolases , alfa-Glucosidases , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/síntese química , alfa-Glucosidases/metabolismo , alfa-Glucosidases/química , Humanos , Conformação Molecular , Relação Estrutura-Atividade , Teoria da Densidade Funcional , CicloexanóisRESUMO
Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi1, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease2-4, and mouse knockout5 and inherited human mutations6 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.
Assuntos
Glucose/biossíntese , Paládio/metabolismo , Biocatálise , Ativação Enzimática , Galactose/metabolismo , Glucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Cinética , Difosfato de Uridina/metabolismoRESUMO
The marine Bacteroidota Zobellia galactanivorans has a polysaccharide utilization locus dedicated to the catabolism of the red algal cell wall galactan carrageenan and its unique and industrially important α-3,6-anhydro-D-galactose (ADG) monosaccharide. Here we present the first analysis of the specific molecular interactions the exo-(α-1,3)-3,6-anhydro-D-galactosidase ZgGH129 uses to cope with the strict steric restrictions imposed by its bicyclic ADG substrate - which is ring flipped relative to D-galactose. Crystallographic snapshots of key catalytic states obtained with the natural substrate and novel chemical tools designed to mimic species along the reaction coordinate, together with quantum mechanics/molecular mechanics (QM/MM) metadynamics methods and kinetic studies, demonstrate a retaining mechanism where the second step is rate limiting. The conformational landscape of the constrained 3,6-anhydro-D-galactopyranose ring proceeds through enzyme glycosylation B1,4 â [E4] â E4/1C4 and deglycosylation E4/1C4 â [E4] â B1,4 itineraries limited to the Southern Hemisphere of the Cremer-Pople sphere. These results demonstrate the conformational changes throughout catalysis in a non-standard, sterically restrained, bicyclic monosaccharide and provide a molecular framework for mechanism-based inhibitor design for anhydro-type carbohydrate-processing enzymes and for future applications involving carrageenan degradation. In addition, it provides a rare example of distinct niche-based conformational itineraries within the same carbohydrate-active enzyme family.
RESUMO
This Account describes the manner whereby nature controls the Fenton-type reaction of O-O homolysis of hydrogen peroxide and harnesses it to carry out various useful oxidative transformations in metalloenzymes. H2O2 acts as the cosubstrate for the heme-dependent peroxidases, P450BM3, P450SPα, P450BSß, and the P450 decarboxylase OleT, as well as the nonheme enzymes HppE and the copper-dependent lytic polysaccharide monooxygenases (LPMOs). Whereas heme peroxidases use the Poulos-Kraut heterolytic mechanism for H2O2 activation, some heme enzymes prefer the alternative Fenton-type mechanism, which produces â¢OH radical intermediates. The fate of the â¢OH radical is controlled by the protein environment, using tight H-bonding networks around H2O2. The so-generated â¢OH radical is constrained by the surrounding H-bonding interactions, the orientation of which is targeted to perform H-abstraction from the Fe(III)-OH group and thereby leading to the formation of the active species, called Compound I (Cpd I), Por+â¢Fe(IV)âO, which performs oxidation of the substrate. Alternatively, for the nonheme HppE enzyme, the O-O homolysis catalyzed by the resting state Fe(II) generates an Fe(III)-OH species that effectively constrains the â¢OH radical species by a tight H-bonding network. The so-formed H-bonded â¢OH radical acts directly as the oxidant, since it is oriented to perform H-abstraction from the C-H bond of the substrate (S)-2-HPP. The Fenton-type H2O2 activation is strongly suggested by computations to occur also in copper-dependent LPMOs and pMMO. In LPMOs, the Cu(I)-catalyzed O-O homolysis of the H2O2 cosubstrate generates an â¢OH radical that abstracts a hydrogen atom from Cu(II)-OH and forms thereby the active species of the enzyme, Cu(II)-Oâ¢. Such Fenton-type O-O activation can be shared by both the O2-dependent activations of LPMOs and pMMOs, in which the O2 cosubstrate may be reduced to H2O2 by external reductants. Our studies show that, generally, the H2O2 activation is highly dependent on the protein environment, as well as on the presence/absence of substrates. Since H2O2 is a highly flexible and hydrophilic molecule, the absence of suitable substrates may lead to unproductive binding or even to the release of H2O2 from the active site, as has been suggested in P450cam and LPMOs, whereas the presence of the substrate seems to play a role in steering a Fenton-type H2O2 activation. In the absence of a substrate, the hydrophilic active site of P450BM3 disfavors the binding and activation of H2O2 and protects thereby the enzyme from the damage by the Fenton reaction. Due to the distinct coordination and reaction environment, the Fenton-type H2O2 activation mechanism by enzymes differs from the reaction in synthetic systems. In nonenzymatic reactions, the H-bonding networks are quite dynamic and flexible and the reactivity of H2O2 is not strategically constrained as in the enzymatic environment. As such, our Account describes the controlled Fenton-type mechanism in metalloenzymes, and the role of the protein environment in constraining the â¢OH radical against oxidative damage, while directing it to perform useful oxidative transformations.
Assuntos
Peróxido de Hidrogênio , Metaloproteínas , Cobre/química , Compostos Férricos , Heme/química , Peróxido de Hidrogênio/química , Metaloproteínas/metabolismo , Oxirredução , Peroxidases/metabolismoRESUMO
Bacterial ß-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as ß-cellulose, ß-glucans and ß-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C1 conformation to a distorted 1 S3 /1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OHâ â â nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.
Assuntos
Glicosídeo Hidrolases , Simulação de Dinâmica Molecular , Glicosídeo Hidrolases/metabolismo , Ligação de Hidrogênio , Conformação Proteica , Açúcares , Especificidade por Substrato , Cristalografia por Raios X , CatáliseRESUMO
UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.
Assuntos
Racemases e Epimerases , Uridina Difosfato Ácido Glucurônico , Uridina Difosfato Ácido Glucurônico/metabolismo , Descarboxilação , Rotação , Ácido Glucurônico , Racemases e Epimerases/metabolismo , Cetoses , NAD/químicaRESUMO
The development of small-molecule covalent inhibitors and probes continuously pushes the rapidly evolving field of chemical biology forward. A key element in these molecular tool compounds is the "electrophilic trap" that allows a covalent linkage with the target enzyme. The reactivity of this entity needs to be well balanced to effectively trap the desired enzyme, while not being attacked by off-target nucleophiles. Here we investigate the intrinsic reactivity of substrates containing a class of widely used electrophilic traps, the three-membered heterocycles with a nitrogen (aziridine), phosphorus (phosphirane), oxygen (epoxide) or sulfur atom (thiirane) as heteroatom. Using quantum chemical approaches, we studied the conformational flexibility and nucleophilic ring opening of a series of model substrates, in which these electrophilic traps are mounted on a cyclohexene scaffold (C6 H10 Y with Y=NH, PH, O, S). It was revealed that the activation energy of the ring opening does not necessarily follow the trend that is expected from C-Y leaving-group bond strength, but steeply decreases from Y=NH, to PH, to O, to S. We illustrate that the HOMONu -LUMOSubstrate interaction is an all-important factor for the observed reactivity. In addition, we show that the activation energy of aziridines and phosphiranes can be tuned far below that of the corresponding epoxides and thiiranes by the addition of proper electron-withdrawing ring substituents. Our results provide mechanistic insights to rationally tune the reactivity of this class of popular electrophilic traps and can guide the experimental design of covalent inhibitors and probes for enzymatic activity.
Assuntos
Aziridinas , Aziridinas/química , Compostos de Epóxi/química , Nitrogênio , Fósforo , Cicloexenos , Enxofre , OxigênioRESUMO
The optical control and investigation of neuronal activity can be achieved and carried out with photoswitchable ligands. Such compounds are designed in a modular fashion, combining a known ligand of the target protein and a photochromic group, as well as an additional electrophilic group for tethered ligands. Such a design strategy can be optimized by including structural data. In addition to experimental structures, computational methods (such as homology modeling, molecular docking, molecular dynamics and enhanced sampling techniques) can provide structural insights to guide photoswitch design and to understand the observed light-regulated effects. This review discusses the application of such structure-based computational methods to photoswitchable ligands targeting voltage- and ligand-gated ion channels. Structural mapping may help identify residues near the ligand binding pocket amenable for mutagenesis and covalent attachment. Modeling of the target protein in a complex with the photoswitchable ligand can shed light on the different activities of the two photoswitch isomers and the effect of site-directed mutations on photoswitch binding, as well as ion channel subtype selectivity. The examples presented here show how the integration of computational modeling with experimental data can greatly facilitate photoswitchable ligand design and optimization. Recent advances in structural biology, both experimental and computational, are expected to further strengthen this rational photopharmacology approach.
Assuntos
Ativação do Canal Iônico/efeitos da radiação , Canais Iônicos/metabolismo , Optogenética/métodos , Animais , Sítios de Ligação , Humanos , Ligantes , Luz , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Processos FotoquímicosRESUMO
Long-range electron transfer (ET) in metalloenzymes is a general and fundamental process governing O2 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 O2 , directly leads to LPMO-CuII -O2- , bypassing the formation of the generally assumed LPMO-CuI species. The uncovered ET mechanism rationalizes experimental observations and might have far-reaching implications for LPMO catalysis as well as the O2 - or CO-binding-enhanced long-range ET processes in other metalloenzymes.
Assuntos
Desidrogenases de Carboidrato/metabolismo , Transporte de Elétrons/fisiologia , Oxigenases de Função Mista/metabolismo , Oxigênio/metabolismo , Polissacarídeos/metabolismo , HumanosRESUMO
The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (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 ß-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This ß-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.
Assuntos
Cicloexanóis/metabolismo , Cisteína/metabolismo , Inibidores Enzimáticos/metabolismo , Glicosídeo Hidrolases/metabolismo , Biocatálise , Cristalografia por Raios X , Cicloexanóis/química , Cicloexanóis/farmacologia , Cisteína/química , Teoria da Densidade Funcional , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/química , Simulação de Dinâmica Molecular , Estrutura MolecularRESUMO
Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-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-1. 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.
Assuntos
Glicosídeo Hidrolases/metabolismo , Fosforilases/metabolismo , Mutação Puntual , Domínio Catalítico , Glicosídeo Hidrolases/genéticaRESUMO
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 A. niger 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.
Assuntos
Ciclopentanos/química , Inibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/análise , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/análise , Aziridinas/síntese química , Aziridinas/química , Basidiomycota/enzimologia , Ciclopentanos/síntese química , Inibidores Enzimáticos/síntese química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Cinética , TermodinâmicaRESUMO
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 Km 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 kcat/Km. 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.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Aldeído Oxirredutases/metabolismo , Células-Tronco Neoplásicas/metabolismo , Retinal Desidrogenase/metabolismo , Tretinoína/metabolismo , Humanos , Magnésio/metabolismo , Modelos Moleculares , Células-Tronco Neoplásicas/patologia , Retinaldeído/metabolismo , Especificidade por SubstratoRESUMO
The mechanism for dioxygen activation represents one of the core issues in metalloenzymes. In most cases, the activation of the O2 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 O2 activation in MPnS is mediated by an adjacent Lysine residue (Lys28) in the active site, leading to an unusual H 2 O 2 intermediate in the reductant-independent nonheme MPnS enzyme. Furthermore, the so-generated H 2 O 2 intermediate is subsequently employed in a Fenton-type reaction, leading to a locked â¢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 2 activation in metalloenzymes.
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SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus ß-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a ß-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a ß-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'ß-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.
Assuntos
Glicosiltransferases/metabolismo , Hidrolases/metabolismo , Engenharia de Proteínas , beta-Glucosidase/metabolismo , Biocatálise , Teoria Quântica , Sulfolobus solfataricus/enzimologiaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Porinas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas putida/metabolismo , Anti-Infecciosos/metabolismo , Proteínas de Bactérias/química , Transporte Biológico , Humanos , Modelos Moleculares , Porinas/química , Conformação Proteica , Pseudomonas putida/química , Especificidade por SubstratoRESUMO
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 photosystemâ 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.
RESUMO
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 Au18(SR)14 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 Au18(SR)14 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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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 O2 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.
Assuntos
Catalase/química , Radicais Livres/química , Metionina/química , Oxigênio/química , Triptofano/química , Tirosina/química , Domínio Catalítico , Transporte de Elétrons , Heme/química , Histidina/química , Simulação de Dinâmica Molecular , Oxirredução , Peroxidases/química , Conformação Proteica , PrótonsRESUMO
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 4 C1 /4 H5 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.