Overexpression, purification, and crystallization of BtrG, a cyclotransferase involved in the last step of the butirosin biosynthesis.

• Grant number: 25/22764-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2025
• End date: October 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Marina Gabriela Lopes Pagiato
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 25/14004-5 - Structural studies of enzymes involved in the butirosin biosynthesis, AP.R

Abstract

The principal therapeutic approach for treating bacterial infections is based on the use of antibacterial agents. One of the difficulties associated with this treatment is the development of resistance mechanisms. The search for new molecules that can circumvent these resistance mechanisms requires a global financial and public health effort. Given the need to develop strategies to overcome antimicrobial resistance, one method is to explore biosynthetic mechanisms in natural products. Among these products, the project focuses on exploring the biosynthesis of butirosin, an aminoglycoside with a structure similar to that of neomycin, produced by the bacterium Niallia circulans. Its structure is reported to evade the action of bacterial resistance mechanisms due to the presence of a substitution at N'1 of its aglycone 2-deoxystreptamine (2-DOS). The (S)-4-amino-2-hydroxybutyrate (AHBA) portion is naturally added in its biosynthesis process and was semi-synthetically incorporated to form amikacin and plazomicin. The route for the formation of the AHBA group, as well as its molecule, is well documented, elucidating the role of the seven enzymes (btrI, btrJ, btrK, btrH, btrG, btrO and btrV) that participate together within the same gene cluster. However, since the production of AHBA does not follow a common pattern, it is likely that its enzymes have specific catalysis, which makes it difficult to apply biotechnological strategies to generate new variations of aminoglycosides through biocatalysis or combinatorial biosynthesis. This project focuses on understanding the structure and molecular mechanisms of the ¿-glutamyl-transferase btrG. To achieve this objective, methodological approaches in the field of molecular biology and biophysics will be employed, with an emphasis on crystallography. In this way, it is hoped that it will be possible to acquire data that has the potential to be applied in biotechnological procedures aimed at manufacturing compounds derived from aminoglycosides that are resistant to EMAs. (AU)