Structure and function of iterative polyketide synthases (iPKSs) and hybrid systems of iPKS and Nonribosomal peptide synthases (iPKS-NRPSs) involved in antibiotic biosynthesis

• Grant number: 24/23287-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: March 01, 2025
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Veronica Silva Valadares
• 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

Among the natural products, polyketides and non-ribosomal peptides are very diverse compounds synthesized by giant enzymes, polyketide synthases (PKSs), and non-ribosomal peptide synthases (NRPS), respectively. Through various iterative elongation cycles, PKSs catalyze the sequential condensation of two or three carbon units to a growing acyl chain covalently tethered to a carrier domain (ACP). NRPSs act similarly to PKSs but add amino acid units instead to a growing polypeptide chain. Type I PKSs are classified into modular or iterative PKSs (iPKSs). iPKSs are usually formed by a single module that performs several or multiple rounds of chain extension. Since PKSs are enzymes responsible for producing a large number of polyketides, researchers and pharmaceutical companies are intensely interested in understanding these enzymes for applications in rational protein engineering to produce new natural product derivatives. iPKSs are poorly understood, and several intriguing characteristics of this enzyme remain unclear. iPKSs have sophisticated and elaborate catalysis since the ¿-reduction set of enzymes is used differently in each iterative round of chain extension. Although several studies have demonstrated the reconstitution of iPKS products in vitro and information has been gathered using genetic validation and mass spectroscopy, the structure and the dynamic of an iPKS remain elusive. Also, little is known about how these enzymes transfer their products to an NRPS or how they control the use of reducing domains. Among the natural products biosynthesized by an iPKS, 6-methyl salicylic acid (6-MSA) was one of the first polyketides to be investigated. 6-MSA is biosynthetically produced by the iPKS 6-methylsalic acid synthase (6-MSAS), which forms a tetramer with a total mass of 750 kDa in solution. 6-MSAS has been mechanistically studied through in vitro biosynthesis reconstitution. Other intriguing PKSs are those fused with NRPS domains (iPKS-NRPS) and generally have a more complicated and intricate architecture. Monomerically, these enzymes are bigger than 6-MSAS since they have more structural domains. Consequently, these enzymes are even less understood than iPKSs. One of the simplest systems of hybrid iPKS-NRPSs is the enzyme TlmB, which is involved in the biosynthesis of the thiotetronate thiolactomycin (TLM). TlmB was predicted to be an iPKS that recruits three propionate units and then catalyzes three cycles of chain elongation. Since there is a lack of knowledge about the structural mechanism of iPKS and iPKS-NRPS, in this project, we aim to apply different techniques of structural biology for the determination of the domain organization from iPKSs and iPKS-NRPSs and the dynamic of the ACP domain. We will use cryo-electron microscopy, protein crystallography, nuclear magnetic resonance, mass spectroscopy, and SAXS to understand and possibly propose strategies for engineering these systems to produce new antibiotics.