RESUMO
We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.
Assuntos
Antibacterianos/química , Escherichia coli O157/efeitos dos fármacos , Toxina Shiga I/antagonistas & inibidores , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Escherichia coli O157/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologia , Componente Amiloide P Sérico/metabolismo , Toxina Shiga I/químicaRESUMO
[reaction: see text] The first example of a multivalent heterofunctional inhibitor-adaptor, called "BAIT", is described. This multivalent inhibitor-adaptor is able to capture a "target" receptor (Shiga toxin) through its recognition of one ligand of a heterobivalent headgroup while the other ligand binds to an endogenous "trap" protein (serum amyloid P component, SAP). BAIT showed markedly enhanced inhibition of toxin activity. An efficient synthesis of this multivalent cluster containing heterobifunctional ligands was accomplished by chemical and chemoenzymatic approaches.
Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Toxina Shiga/antagonistas & inibidores , Toxina Shiga/química , Glicosilação , Ligantes , Estrutura Molecular , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/metabolismoRESUMO
The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid-base catalytic residue. AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.
Assuntos
Domínio Catalítico/fisiologia , Glicosídeo Hidrolases/química , Xilose/análogos & derivados , Ácidos/química , Álcalis/química , Azidas/química , Bacillus , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/fisiologia , Clonagem Molecular , Glicosídeo Hidrolases/genética , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectroscopia de Infravermelho com Transformada de Fourier , Xilose/químicaRESUMO
[reaction: see text] Tuning the reactivity of glycosyl donors derived from 2-amino-2-deoxy glucose by selective introduction of different N-protecting (NPhth and NHTroc) and anomeric leaving groups (ethylthio and phenylthio) enabled highly efficient oligosaccharide synthesis in a one-pot manner. One-pot sequential glycosylation of three and four units of 2-amino-2-deoxy glucose gave trisaccharides and tetrasaccharide in 50-81% yields.
Assuntos
Glucosamina/síntese química , Oligossacarídeos/síntese química , Glucosamina/química , Tioglicosídeos/químicaAssuntos
Entropia , Componente Amiloide P Sérico/antagonistas & inibidores , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Trissacarídeos , Animais , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Morte Celular/efeitos dos fármacos , Chlorocebus aethiops , Desenho de Fármacos , Glicerol/química , Humanos , Ligantes , Substâncias Macromoleculares/antagonistas & inibidores , Substâncias Macromoleculares/química , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Piruvatos/química , Componente Amiloide P Sérico/química , Toxina Shiga I/química , Toxina Shiga II/química , Trissacarídeos/síntese química , Trissacarídeos/química , Trissacarídeos/farmacologia , Células VeroRESUMO
beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Brønsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.
Assuntos
Bacillaceae/enzimologia , Xilosidases/química , Substituição de Aminoácidos , Bacillaceae/genética , Sequência de Bases , Domínio Catalítico/genética , DNA Bacteriano/genética , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Azida Sódica , Especificidade por Substrato , Xilosidases/genética , Xilosidases/metabolismoRESUMO
alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant. The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.