Structural study of critical enzymes involved in the Butirosin biosynthesis

• Grant number: 22/13839-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: April 01, 2023
• End date: September 30, 2026
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Renan Passos Freire
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Bacterial resistance against antimicrobials is a serious public health problem, limiting the treatment of infections to only a few molecules of last resort which often have severe side effects. Among the classes used to treat infections, there are aminoglycosides, which include antibiotics such as neomycin, gentamicin, and kanamycin. Resistance against these antibiotics, particularly by the aminoglycoside-modifying enzymes (AMEs), has also been consistently reported. Interestingly, butirosin, an aminoglycoside with a similar structure to neomycin, and produced by Niallia circulans, shows insensitivity to the action of AMEs due to the presence of substitution in its 2-deoxy-streptamine aglycone (2-DOS) for the addition of an (S)-4-amino-2-hydroxybutyrate (AHBA) moiety. The importance of the presence of the AHBA group can be demonstrated by the semi-synthetic incorporation of this group into kanamycin to produce amikacin, which should be currently the most commercialized aminoglycoside. The biosynthetic route for AHBA moiety in butirosin is well understood, including the function of all enzymes involved in the biosynthesis of this group. However, since the AHBA biosynthesis is unusual, the enzymes must also have unusual structures and catalytic mechanisms. The lack of understanding about these enzymes limits the application of biotechnological strategies in the production of new aminoglycoside derivatives through biocatalysis or combinatorial biosynthesis. Thus, this project focuses on understanding the molecular structures and mechanisms of enzymes involved in key steps of butirosin biosynthesis, particularly of the AHBA group. The practical methods used in this project involve the field of molecular biology and biophysics, particularly protein crystallography, obtaining structures of key enzymes in their apo form and/or in the presence of ligands. We aim to understand unusual catalysis mechanisms and generate information that might be useful in biotechnological processes to produce EMA-insensitive aminoglycoside-derived molecules.