OVEREXPRESSION, PURIFICATION, AND CRYSTALLIZATION OF BTRG, THE CYCLOTRANSFERASE RESPONSIBLE FOR THE FINAL STEP OF BUTIROSIN BIOSYNTHESIS

Bacterial resistance to aminoglycosides represents a major public health challenge, driving the search for novel antibiotics capable of overcoming resistance mechanisms. Butirosin, an aminoglycoside produced by Niallia circulans, naturally contains the (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, a structural modification that reduces the susceptibility of the antibiotic to aminoglycoside-modifying enzymes. This structural feature was later exploited in the development of semisynthetic aminoglycosides, such as amikacin and plazomicin. In the final step of butirosin biosynthesis, the γ-glutamyl cyclotransferase BtrG catalyzes the removal of the γ-glutamyl protecting group from AHBA. However, the relationship between the structure of BtrG and its catalytic function remains poorly understood. This study aims to obtain a BtrG sample suitable for structural characterization by X-ray crystallography. Initially, the recombinant protein was heterologously expressed in different Escherichia coli strains, including BL21 (DE3), Rosetta, Star, and strains co-expressing the molecular chaperones GroES/GroEL through the pGro7 plasmid. The proteins were purified by immobilized metal affinity chromatography (IMAC) and by molecular size exclusion. However, the initial constructs exhibited low recovery of soluble protein, limiting the production of samples suitable for structural studies. Based on structural predictions generated by AlphaFold3, which indicated low confidence in the N- and C-terminal regions of the protein, two complementary experimental strategies were pursued: cloning of the GGCT catalytic domain to improve protein stability and solubility, and cloning of the full-length btrG gene to enable comparison between the full-length protein and the isolated catalytic domain. The constructs were confirmed by DNA sequencing and subjected to heterologous expression and affinity purification assays. The results indicate that further optimization of protein expression and stabilization is required, as protein precipitation was observed during the concentration step, prompting the evaluation of alternative purification conditions, including buffers supplemented with glycerol. The results obtained establish a strategy for producing a structurally stable BtrG sample, an essential step toward determining its three-dimensional structure, elucidating its catalytic mechanism, and supporting future enzyme engineering and combinatorial biosynthesis approaches for the development of novel aminoglycosides capable of overcoming bacterial resistance.