Use of site-directed mutagenesis in the understanding of the catalytic domain activity of the human SDH enzyme

• Grant number: 14/27019-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2015
• End date: February 29, 2016
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Paulo Arruda
• Grantee: Lucas Vale Adjafre
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

The Saccharopine pathway is the main degradation pathway of Lysine in plants and animals. The first gene of the pathway is the AASS, which encodes the bifunctional enzyme Aminoadipate-Semialdehyde Synthase, responsible for the convertion of Lysine in Aminoadipate Semialdehyde (AASA) via the intermediate Saccharopine. It is then oxidized to Aminoadipic Acid by the Aminoadipate Semialdehyde Desidrogenase (ALDH7A1). A defect in this enzyme causes Piridoxine Dependent Epilepsy (PDE) which is mainly caused by the accumululation of AASA and its cyclic form piperideine-6-carboxylate (P6C) in the organism. Recently, our group was responsible for expressing, purifying and resolving the crystalographic structure of the Saccharopine Desidrogenase (SDH) of the human enzyme AASS. The structural knowledge can be used for better understanding of the biochemical operation of this enzyme. The current project aims, via site-directed mutagenesis, the ponctual substitution by alanine of some aminoacids, which were chosen by computational biology for a better understanding of the catalytic sites and ligation points of the enzyme to the saccharopine.