Structural study of glycosyltransferases dependent on P450-type helper proteins involved in the biosynthesis of macrolide antibiotics

• Grant number: 24/21568-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: May 01, 2025
• End date: December 31, 2029
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Luís Victor Santana Rosa
• 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

Bacterial resistance to antibiotics is one of the greatest public health challenges today, characterized as a growing threat that deteriorates the efficacy of these drugs. Natural products from microorganisms have shown promise in the search for new active molecules that contain mechanisms capable of circumventing this resistance. Among natural products, polyketides represent a broad class of structurally diverse compounds whose biological activity is often related to the functional groups that are attached to their central skeleton (aglycone). Macrolides represent a class of widely used antibiotics and are an example of polyketides whose activity is dependent on sugar molecules. The enzymes that perform glycosylation of polyketides are glycosyltransferases (GTs), which have a strict specificity for 6-deoxysugars, but a relaxed specificity for unusual sugars and acceptor substrates. Exploiting this catalytic flexibility of GTs from natural products may contribute to the generation of new compounds through glycodiversification. In turn, these new compounds may exhibit novel biological activities and improved pharmacokinetic properties. In addition to relaxed specificity, there is a small group of GTs that exhibit a peculiar behavior in which an auxiliary protein (AP) is required for their catalytic activity. Studying the interaction and conformational changes that occur during the formation of the glycosyltransferase-auxiliary protein complex is essential to understand the influence that these APs exert on GTs and, thus, generate information that may be useful in the application of glycodiversification. The target GT/AP pairs that we propose in this study are: EryCIII/EryCII from Saccharopolyspora erythrea; MycB/MydC from Micromonospora griseorubida; DesVII/DesVIII from Streptomyces venezuelae, TylM2/TylM3 from Streptomyces fradiae, AngMII/AngMIII from Streptomyces eurythermus, Srm5/Srm6 and Srm29/Srm28 from Streptomyces ambofaciens. The constructs containing the genes for these proteins will be expressed in competent E. coli strains, purified by liquid chromatography techniques and then subjected to structural crystallography or cryo-electron microscopy assays, as well as biophysical tests of isothermal titration calorimetry and surface plasmon resonance to identify the formation constants of the protein complexes. Thus, this project aims to produce knowledge in order to promote advances in the understanding of this unusual family of GTs that require an auxiliary protein for their activity and also contribute to future work in synthetic biology or glycodiversification.