Estudo das bases moleculares da especificidade pelo substrato de uma beta-glicosidase (AF052729) de Spodoptera frugiperda / Study of the molecular basis of substrate specificity in a Spodoptera frugiperda beta-glycosidase (AF052729)

AUTOR(ES)

Julio Henrique Kravcuks Rozenfeld

DATA DE PUBLICAÇÃO

2006

RESUMO

The Mr 50,000 Spodoptera frugiperda beta-glycosidase (Sfgli50) has active site amino acid residues that interact non-covalently with the substrate. These non-covalent interactions determine the enzyme’s specificity towards its substrates. This work aims to study the role of these amino acids and its interactions with the substrate in the specificity of Sfgli50. A specificity prediction model was created for such purpose. This model is based on interaction energies between different amino acid residues in positions 451 and 39 of the enzyme and hydroxyls 4 and 6 of the substrates glycone. The production and characterization of three double-mutants (S451N39, S451E39 and A451E39) were used to test the model, which proved to be qualitatively appropriate to predict the Sfgli50 specificity when comparing glucosides to galactosides. However, the model failed to predict the differences in specificity between fucosides and galactosides. These results indicate that the models assumptions of independent interactions with the substrate and structural conservation of the active site in the mutants were wrong. Sfgli50 glycone residues H142 and N186, whose interaction energies had not been previously determined, were also investigated. Site-directed mutagenesis replaced the former residues in positions 142 and 186 for Alanine. The mutant proteins H142A and N186A were synthesized in bacteria and partially purified. Mutant N186A presented low catalytic activity, hindering its characterization. In contrast, mutant H142A is less specific than the wild-type Sfgli50. Kinetic parameters indicated that it is specially less specific to fucosides.

ASSUNTO(S)

spodoptera frugiperda bioquímica enzyme biochemistry enzima spodoptera frugiperda

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