Structural and functional studies of key enzymes involved in the apramycin biosynthesis.

• Grant number: 24/16330-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: June 01, 2025
• End date: December 31, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Guilherme Henrique Aparecido de Oliveira
• 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

Aminoglycosides, one of the groups of bioactive molecules isolated from actinobacteria, are antibiotics rich in amino and hydroxyl groups. The mechanism of action of these molecules consists of the interaction of the amino groups contained in their amino sugars with the ribosomal RNA of bacteria. The binding occurs specifically at the A binding site of the 30S portion of the bacterial ribosome, leading to incorrect reading of the ribosomal RNA and consequently the synthesis of truncated proteins or proteins with incorrect sequences. Apramycin, on the other hand, is a peculiar aminoglycoside due to the presence of an octose ring. This makes this molecule insensitive to most aminoglycoside resistance mechanisms, especially by aminoglycoside-modifying enzymes (AMEs). However, most of the enzymes involved in the biosynthesis of apramycin, including those involved in forming the octose ring, do not have their three-dimensional structure elucidated or their catalysis mechanisms understood. Therefore, this project aims to obtain the structures, substrate or analog recognition mechanisms, and possibly the catalytic mechanisms of key and unique enzymes in apramycin biosynthesis. To achieve our objectives, the proteins will be expressed heterologously and purified to ensure a high level of purity in order to obtain crystals with possible ligands for the purpose of subsequent diffraction at the national synchrotron light laboratory and structure resolution using computational methods. The resolution of these structures, especially AprG, AprP and AprI, as well as the possible unraveling of the catalytic system of these enzymes involved in the unique central structure of apramycin, makes it possible to understand the synthesis of this molecule, facilitating future work in the engineering of second-generation antibiotics with apramycin as a base.