Full text
| |
| Author(s): |
Priscila dos Santos Bury
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2019-09-06 |
| Examining board members: |
Marcio Vinícius Bertacine Dias;
Felipe Santiago Chambergo Alcalde;
Cristiane Rodrigues Guzzo Carvalho;
Fábio Cesar Gozzo
|
| Advisor: | Marcio Vinícius Bertacine Dias |
| Abstract | |
Aminoglycoside is a class of antibiotics characterized to contain amino sugars linked through glycosidic linkage to a central aminocyclitol ring. These antibiotics are primarily used to treat infections caused by gram-negative aerobic bacteria. Its action mechanism consists in the inhibition of protein synthesis binding to the 30S subunit of the bacterial ribosome. Unfortunately, however, clinical use of these molecules is limited due to otoxicity and nephrotoxicity. Aminoglycosides include tobramycin, amikacin, kanamycin, streptomycin, paromycin, neomycin, gentamicin and sisomicin. Sisomicin and gentamicin have the 2-deoxystreptamine as the central ring and are highly aminated and methylated molecules. Thus, the biosynthetic route of these molecules have several enzymes that perform such decorations on their rings, and that are biotechnological targets to produce new aminoglycoside derivatives. In this work, we used heterologous expression methods of proteins, affinity chromatography and molecular exclusion, and X-ray crystallography for structural determination of enzymes involved in the biosynthesis of gentamicin and sisomicin. Thus, the crystal structures of the following pyridoxal-5\'-phosphate dependent enzymes were obtained: Sis5, GenB2, GenB3 and GenB4 in binary complex with their cofactors and ternary complex with biosynthesis intermediates. Using these structures, details were revealed regarding the shape and mechanism of the enzymes. Besides the peculiarities of each one, wh that allow them to make different modifications in the substrates. In addition, we have obtained structure of the methyltransferase GenN in complex with its substrate and other analogues or alternative substrates. These protein-ligand complexes demonstrated the ability of this enzyme to methylate other aminoglycosides, producing methylated kanamycin and tobramycin, which lack such natural modification, producing non-natural natural products. By obtaining the three-dimensional structures of these enzymes and a deep structural analysis it was possible to carry out a detailed study on the mechanism of catalysis and the promiscuity of such enzymes, demonstrating their potential use in synthetic biology to produce aminoglycoside derivatives. (AU) | |
| FAPESP's process: | 14/07843-6 - Structural analysis of key enzymes for gentamicin and sisomicin biosynthesis |
| Grantee: | Priscila dos Santos Bury |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |