STRUCTURAL ELUCIDATION AND NUCLEOTIDE BINDING STUDIES OF APRK, AN ENZYME INVOLVED IN APRAMYCIN BIOSYNTHESIS

Apramycin is an aminoglycoside distinguished by its unique octose ring, which enables it to evade most aminoglycoside-modifying enzymes. In the apramycin biosynthesis, AprK is a key enzyme,  and its role is to catalyze a reaction in which a nucleotidyl group is transferred to a glucose molecule, resulting in the formation of NDP-β-D-glucose. AprK remains structurally uncharacterized, which limits the understanding of the nucleotide transfer mechanism during apramycin biosynthesis. Here, we preliminarily determine the three-dimensional structure of AprK and characterize its interaction with nucleotide ligands. AprK was heterologously expressed in E. coli BL21(DE3) co-expressing GroEL/GroES chaperones and purified by IMAC followed by SEC. The enzyme was successfully purified as a dimer, and a differential scanning fluorimetry assay was performed to determine the best buffering conditions. AprK crystals were obtained in the presence of ADP, and their X-ray diffraction data were collected at the MANACÁ beamline in Campinas, Brazil, with a resolution up to 2.14 Å (crystal structure refinement is ongoing). The AprK structure displayed a Rossmann-like α/β fold with five α-helices and five β-strands per protomer. The ADP molecule was observed in the putative nucleotide-binding site, interacting with the conserved T/HXGH motif. The bound ADP defines the nucleotide-binding site and provides an initial framework for understanding AprK substrate recognition during apramycin biosynthesis. This architecture is consistent with previously characterized members of the nucleotidyltransferase superfamily.  Additionally, isothermal titration calorimetry (ITC) assays were conducted with different nucleotides, including AMP, TMP, GMP, and UMP. ITC experiments indicated that UMP exhibited the highest affinity (KD = 40.3 μM), whereas TMP bound weakly (KD = 317 μM), suggesting a preference for uridine nucleotides. The preference for UMP over the other nucleotides suggests that AprK exhibits selective nucleotide recognition, which may contribute to substrate specificity during NDP-β-D-glucose biosynthesis.  These findings provide the first structural and biophysical insights into the AprK catalytic mechanism, contributing to the understanding of nucleotide recognition during apramycin biosynthesis.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo  (FAPESP 2024/16330-4) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 88887.933247/2024-00).