RESUMO
Staphylococcus aureus is an important cause of resistant healthcare-associated infections. It has been shown that the wall teichoic acid (WTA) may be an important drug target acting on antibiotic-resistant cells. The UDP-N-acetylglucosamine 2-epimerase, MnaA, is one of the first enzymes on the pathway for the biosynthesis of the WTA. Here, detailed molecular dynamics simulations of S. aureus MnaA were used to characterize the conformational changes that occur in the presence of UDP and UDP-GlcNac and also the energetic landscape associated with these changes. Using different simulation techniques, such as ABMD and GAMD, it was possible to assess the energetic profile for the protein with and without ligands in its active site. We found that there is a dynamic energy landscape that has its minimum changed by the presence of the ligands, with a closed structure of the enzyme being more frequently observed for the bound state while the unbound enzyme favors an opened conformation. Further structural analysis indicated that positively charged amino acids associated with UDP and UDP-GlcNac interactions play a major role in the enzyme opening movement. Finally, the energy landscape profiled in this work provides important conclusions for the design of inhibitor candidates targeting S. aureus MnaA.
Assuntos
Infecções Estafilocócicas/enzimologia , Staphylococcus aureus/enzimologia , Ácidos Teicoicos/química , Sequência de Aminoácidos , Aminoácidos/química , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Carboidratos Epimerases/ultraestrutura , Domínio Catalítico/efeitos dos fármacos , Parede Celular/enzimologia , Farmacorresistência Bacteriana/genética , Metabolismo Energético/genética , Glucosamina/análogos & derivados , Glucosamina/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Difosfato de Uridina/químicaRESUMO
Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.
Assuntos
Proteínas de Bactérias/química , Cisteína Proteases/química , Modelos Moleculares , Xylella/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação Puntual , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Difosfato de Uridina/química , Difosfato de Uridina/metabolismoRESUMO
Xanthomonas campestris GumK (beta-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthesis. No x-ray structures have been reported for this family. To better understand the mechanism of action of the bacterial glycosyltransferases in this family, the x-ray crystal structure of apo-GumK was solved at 1.9 angstroms resolution. The enzyme has two well defined Rossmann domains with a catalytic cleft between them, which is a typical feature of the glycosyltransferase B superfamily. Additionally, the crystal structure of GumK complexed with UDP was solved at 2.28 angstroms resolution. We identified a number of catalytically important residues, including Asp157, which serves as the general base in the transfer reaction. Residues Met231, Met273, Glu272, Tyr292, Met306, Lys307, and Gln310 interact with UDP, and mutation of these residues affected protein activity both in vitro and in vivo. The biological and structural data reported here shed light on the molecular basis for donor and acceptor selectivity in this glycosyltransferase family. These results also provide a rationale to obtain new polysaccharides by varying residues in the conserved alpha/beta/alpha structural motif of GumK.