Molecular basis for conformational changes and role of amino acid residues in the soluble intestinal trehalase from Spodoptera frugiperda larvae.

• Grant number: 12/09218-6
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: December 01, 2012
• End date: April 30, 2016
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Clelia Ferreira Terra
• Grantee: Walciane da Silva
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 11/51685-8 - Insect digestion: a molecular, cellular, physiological and evolutionary approach, AP.TEM

Abstract

Trehalases are important enzymes in insect, fungi and nematodes, groups that cause harm to human beings. This enzyme class have devoted few attention, perhaps due to their secundary role in mammals. Nevertheless, this caracteristic points to trehalases as good targets for pest control.The best known trehalases from the enzymologic point of view are insect midgut enzymes, mainly the soluble trehalase from Spodoptera frugiperda midgut. In this enzyme we disclosed the role of some residues in the active site and amassed evidence that the molecule undergoes substantial conformational changes. By molecular modeling of this enzyme with the one from Escherichia coli ( the only trehalase with resolved 3D structure) we could find regions without defined structure that probably are the more mobile ones and the residues present in the active site. In this project we intend to identify regions or residues responsible for the conformational changes by deleting parts of the molecule with no defined structure and detecting changes in tryptophan fluorescence supression by acrylamide. Such studies will show molecules that cause trehalase conformational changes, as well as which parts of the proteins are responsible for these changes and perhaps we could molecular rendering the trehalase less mobile that can be used in the future to obtain cystals.We also intend to detect the role of certain amino acid residues in the enzyme activity by checking the effect of site directed mutagenesis on the Km, kcat and pKas values.Since trehalase is the precursor in chitin syntheses and a decrease in chitin content was found after supression of trelalase expression, we will test peritrophic membrane functions after using trehalase interfering RNA.