| Congresso Brasileiro de Microbiologia 2023 | Resumo: 893-1 | ||||
Resumo: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 some 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 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 related enzymes must also have unusual structures and catalytic mechanisms. Here in, we are focusing on understanding the molecular structures and mechanisms of enzymes involved in key steps of butirosin biosynthesis, particularly of the AHBA moiety. The practical methods used in this project involve the field of molecular biology and biophysics, particularly protein crystallography, aiming to obtain structures of key enzymes in their apo form and/or in the presence of ligands such as substrate analogs. Nevertheless, bioinformatics approaches play a fundamental role for conducting the data of this project. Four enzymes (btrI, btrH, btrJ and btrG) of this route were cloned into pET28(+) plasmid and recombinant expressions were conducted at BL21 (DE3) and Rosetta (DE3) cells through induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). Purification steps involved firstly an immobilized metal affinity chromatography, with Nickel, followed by a size exclusion chromatography. Several methodologies for quantification (specially molar extinction coefficient and Bradford assay) shows a recovery of 1 - 30 mg.ml-1 recombinant protein. At this moment, one particular enzyme, btrH, crystallized at very tiny rod forms, showing that optimization of crystallization conditions could enhance the formation of large crystals. New strategies for solving the structures of the most enzymes will be explored such as RMN and Cryo-EM. We aim to understand unusual catalytic mechanisms and generate information that might be useful in biotechnological processes to produce AME-insensitive aminoglycoside-derived molecules. Applying this knowledge into the field of combinatorial biosynthesis relief at the development of new molecules. Palavras-chave: Aminoglycoside, Antibiotic resistance, Structural biology, Natural products Agência de fomento:Fundação de Amparo a Pesquisa do Estado de São Paulo | |||||