Structural characterization of iterative polyketide synthase and epoxidases from the biosynthesis of natural products

• Grant number: 23/01116-4
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: August 01, 2023
• End date: July 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Gabriel Stephani de Oliveira
• Supervisor: Ben Francesco Luisi
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Cambridge, England
• Associated to the scholarship: 17/23627-0 - Determination of the structural bases of Biosynthetic formation of heterocyclic rings in ionophores polyethers, BP.PD

Abstract

Natural products represent an important source of bioactive materials for medical and veterinary use. Although the field of biological chemistry has had considerable advances in knowledge of these compounds, there is still a lack of understanding of the underlying mechanisms of their biogenesis. An important class of natural products includes polyketides, which are molecules that have distinctive mechanisms of biosynthesis. Polyketides are a very diverse class of compounds synthesized by giant enzymes, the polyketide synthases (PKSs). Type I PKS are further classified into modular or iterative PKS (iPKS). Modular PKSs are organized in protein modules forming multi-enzymatic polypeptides to create an assembly line where each module is responsible to condense a single unit of two or three carbons in the growing polyene. In contrast, iPKSs are usually formed by a single module that performs multiple rounds of chain extension. After the production of the polyene chain by PKSs, several tailoring enzymes carry out modifications to generate the bioactive compound, such as adding chemical groups or catalyzing additional cyclization reactions. In this project, we aim to study different events during the biosynthesis of two groups of polyketides, 6-methyl salicylic acid (6-MSA) and polyether ionophores (PEIs). 6-MSA, an important natural product with anticancer activity, is biosynthesized by the iPKS 6-methylsalic acid synthase (6-MSAS). This megasynthase forms a tetramer with a total mass of 750 kDa. We aim to solve the structure of this iPKS and obtain insights into the domain organization and molecular mechanisms for this class of enzymes. To explore the biosynthesis of PEIs, we aim to study the mechanism of formation of the tetrahydrofuran and tetrahydropyran rings, with special attention to the epoxidases, which catalyze an epoxidation in the first step, and to the epoxide hydrolases, which catalyse the sequential opening and cyclization to cyclic ethers in the polyene. To achieve our objectives, in this project we will employ cryo-electron microscopy (Cryo-EM) to structurally characterize iPKSs systems and crystallography or Cryo-EM for epoxidases aiming to understand their enzymatic mechanism. We also aim to study the structure of these enzymes in complex with substrate analogs and/or products that will be provided by our collaborating research groups. These enzymatic events are still underexplored and have increasingly attracted scientific attention because of their applicability in synthetic biology or biocatalysis to produce new bioactive compounds. (AU)