Cloning, expression and biochemical characterization of 3-methylglutaconyl-CoA hydratase of Trypanosoma brucei

• Grant number: 15/09702-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2015
• End date: December 31, 2015
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Sergio Schenkman
• Grantee: Daniela Fernández Curado
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Protozoan parasites of the order Kinetoplastida use amino acids as a carbon source. Therefore, the characterization of amino acid catabolism pathways is important in understanding how the different amino acids are used by these organisms. Based on genome analysis In Trypanosoma brucei are found 8 enzymes with large similarity to enzymes acting on different substrates containing acyl- Coenzyme A (crotonases), one of which could be the enzyme responsible for hydration of 3-Methyl-glutaconyl CoA, penultimate enzyme in leucine catabolic pathway. In this project we intend to identify the enzyme involved in this step by cloning gene fragments predicted for the possible candidate genes to encode 3- MGCoA hydratases. We intend to express them in Escherichia coli. Obtaining of recombinant proteins will determine the enzyme responsible for this step of the pathway and allow us to characterize some of its biochemical and structural properties.