RESUMO
Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and ß-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.
Assuntos
Proteínas de Bactérias , Glicosiltransferases , Lipoproteínas , Staphylococcus aureus , Ácidos Teicoicos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Staphylococcus aureus/metabolismo , Especificidade por Substrato , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Difosfato de Uridina/metabolismoRESUMO
The widespread and uncontrollable emergence of antibiotic-resistant bacteria, especially methicillin-resistant Staphylococcus aureus, has promoted a wave of efforts to discover a new generation of antibiotics that prevent or treat bacterial infections neither as bactericides nor bacteriostats. Due to its crucial role in virulence and its nonessentiality in bacterial survival, sortase A has been considered as a great target for new antibiotics. Sortase A inhibitors have emerged as promising alternative antivirulence agents against bacteria. Herein, the structural and preparative aspects of some small synthetic organic compounds that block the pathogenic action of sortase A have been described.
RESUMO
Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.
Assuntos
Actinomyces/crescimento & desenvolvimento , Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Biofilmes/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Actinomyces/efeitos dos fármacos , Actinomyces/enzimologia , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Células CultivadasRESUMO
Staphylococcus aureus is a leading cause of hospital-acquired infections in the USA and is a major health concern as methicillin-resistant S. aureus and other antibiotic-resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti-infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure-based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone-based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second-generation analogues that are ~70-fold more potent than the lead molecule. These optimized molecules exhibit broad-spectrum activity against other types of class A sortases, have reduced cytotoxicity, and impair SrtA-mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogues are attractive candidates for further development into anti-infective agents, and highlights the utility of employing NMR spectroscopy and solubility-optimized small molecules in structure-based drug discovery.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Piridazinas/química , Staphylococcus aureus/enzimologia , Aminoaciltransferases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Simulação de Acoplamento Molecular , Piridazinas/farmacologia , Piridazinas/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a "lid", a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the "lid" in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. These results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmounted by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Mutação Puntual , Streptococcus pneumoniae/química , Motivos de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Streptococcus pneumoniae/enzimologia , Especificidade por Substrato , TermodinâmicaRESUMO
The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A ((Ba)SrtA) enzyme. Previously, we discovered that the class A (Ba)SrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of (Ba)SrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (ß7/ß8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II.
Assuntos
Aminoaciltransferases/química , Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Sinais Direcionadores de Proteínas , Aminoaciltransferases/metabolismo , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu(224) and the side chain of Arg(233) form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.
Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Simulação de Dinâmica Molecular , Oxigênio/química , Oxigênio/metabolismo , Staphylococcus aureus/enzimologia , Arginina , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Fímbrias Bacterianas/enzimologia , Ligação de Hidrogênio , Sinais Direcionadores de Proteínas , Staphylococcus aureus/citologiaRESUMO
Staphylococcus aureus is the leading cause of hospital-acquired infections in the United States. The emergence of multidrug-resistant strains of S. aureus has created an urgent need for new antibiotics. Staphylococcus aureus uses the sortase A enzyme to display surface virulence factors suggesting that compounds that inhibit its activity will function as potent anti-infective agents. Here, we report the identification of several inhibitors of sortase A using virtual screening methods that employ the relaxed complex scheme, an advanced computer-docking methodology that accounts for protein receptor flexibility. Experimental testing validates that several compounds identified in the screen inhibit the activity of sortase A. A lead compound based on the 2-phenyl-2,3-dihydro-1H-perimidine scaffold is particularly promising, and its binding mechanism was further investigated using molecular dynamics simulations and conducting preliminary structure-activity relationship studies.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Staphylococcus aureus/enzimologia , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-infective agents as this enzyme attaches virulence factors to the cell wall. Using high-throughput screening, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure-activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione analogs that inhibit SrtA with IC(50) values in the sub-micromolar range. Many of these molecules also inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase inhibitors.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Piridazinas/química , Piridazinas/farmacologia , Staphylococcus aureus/enzimologia , Cisteína Endopeptidases , Descoberta de Drogas , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
Organic molecules possessing intramolecular charge-transfer properties (D-pi-A type molecules) are of key interest particularly in the development of new optoelectronic materials as well as photoinduced magnetism. One such class of D-pi-A molecules that is of particular interest contains photoswitchable intramolecular charge-transfer states via a photoisomerizable pi-system linking the donor and acceptor groups. Here we report the photophysical and electronic properties of the trans to cis isomerization of 1-(pyridin-4-yl)-2-(N-methylpyrrol-2-yl)ethene ligand (mepepy) in aqueous solution using photoacoustic calorimetry (PAC) and theoretical methods. Density functional theory (DFT) calculations demonstrate a global energy difference between cis and trans isomers of mepepy to be 8 kcal mol(-1), while a slightly lower energy is observed between the local minima for the trans and cis isomers (7 kcal mol(-1)). Interestingly, the trans isomer appears to exhibit two ground-state minima separated by an energy barrier of approximately 9 kcal mol(-1). Results from the PAC studies indicate that the trans to cis isomerization results in a negligible volume change (0.9 +/- 0.4 mL mol(-1)) and an enthalpy change of 18 +/- 3 kcal mol(-1). The fact that the acoustic waves associated with the trans to cis transition of mepepy overlap in frequency with those of a calorimetric reference implies that the conformational transition occurs faster than the approximately 50 ns response time of the acoustic detector. Comparison of the experimental results with theoretical studies provide evidence for a mechanism in which the trans to cis isomerization of mepepy results in the loss of a hydrogen bond between a water molecule and the pyridine ring of mepepy.
RESUMO
We report herein the development of a general and mild protocol of oxygen-promoted Pd(II) catalysis resulting in the selective cross-couplings of alkenyl- and arylboron compounds with various olefins. Unlike most cross-coupling reactions, this new methodology works well even in the absence of bases, consequently averting undesired homo-couplings. Nitrogen-based ligands including dimethyl-phenanathroline enhance reactivities and offer a highly efficient and stereoselective methodology to overcome challenging substrate limitations. For instance, oxidative palladium(II) catalysis is effective with highly substituted alkenes and cyclic alkenes, which are known to be incompatible with other known catalytic conditions. Most examined reactions progressed smoothly to completion at low temperatures and in short times. These interesting results provide mechanistic insights and utilities for a new paradigm of palladium catalytic cycles without bases.
Assuntos
Carbono/química , Reagentes de Ligações Cruzadas/química , Nitrogênio/química , Paládio/química , Alcenos/química , Boro/química , Ácidos Borônicos/química , Catálise , Cicloexanonas/química , Esterificação , Ligantes , Estrutura Molecular , Oxirredução , Solventes , TemperaturaRESUMO
[reaction: see text] Oxygen-promoted Pd(II) catalysis facilitated the synthesis of conjugated dienes by cross-coupling of alkenylboronic compounds and various olefins including highly substituted alkenes and cyclohexenone. Under mild conditions, these versatile reactions were efficient and highly stereoselective.