Determination of the structural bases of the cyclic ethers formation mechanism by epoxide hydrolases in the biosynthesis of ionophore polyetheres

• Grant number: 25/06485-3
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2025
• End date: July 31, 2027
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Camilla Cristhina Escobar Pereira
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Natural products constitute a rich source of bioactive compounds used in the treatment of various diseases. However, the molecular mechanisms of heterocyclic ring formation in these compounds remain unclear. In this project, we aim to deepen our understanding of the enzymatic structural mechanisms for the construction of cyclic ethers found in polyether ionophores, particularly nigericin and salinomycin. Analysis of the gene clusters involved in the biosynthesis of these antibiotics revealed the presence of enzymes that catalyze the hydrolysis of epoxide groups, converting them into corresponding diols, with subsequent cyclization to form cyclic ethers. Thus, we propose investigating epoxide hydrolases capable of forming NigBI/NigBII and SalBI/SalBII heterodimers, which are associated with the biosynthesis of nigericin and salinomycin, respectively. For the development of this project, the coding regions for these enzymes have already been subcloned into expression vectors in our laboratory. However, to obtain structures compatible with the expected heterodimers, we will construct chimera proteins by fusing NigBI with NigBII and SalBI with SalBII. To obtain all the proteins involved in the project, we will overexpress them heterologously and purify them using immobilized metal ion affinity chromatography and molecular exclusion chromatography. Subsequently, the different epoxide hydrolases and chimeras will be crystallized in their apo forms and/or in the presence of specific ligands. This will allow the determination and analysis of their structures, contributing to the understanding of their conformations and catalytic mechanisms.