Structural Superimposition

Abstact

Apramycin biosynthesis features an unusual O-5 phosphorylation on octose-containing pseudotrisaccharide intermediates that functions as a pathway recognition tag. AprI catalyzes 7'-N-methylation of the phosphorylated intermediate. Unexpectedly, sequence and phylogenetic analysis place AprI within the HemK/PrmC superfamily of protein-targeting methyltransferases rather than among canonical small-molecule N-methyltransferases. Here, we report the crystal structures of AprI in apo and SAH-bound forms, revealing a clasp-like homodimer formed by the N-terminal region and a highly dynamic substrate-proximal loop. Structure-guided docking, molecular dynamics simulations, and mutational analysis show that the homodimer forms a composite, positively charged pocket for recognition of the O-5 phosphate tag, whereas the flexible loop positions the 7'-amino group of the pseudotrisaccharide substrate for methyl transfer. Together, these findings establish AprI as an unusual PrmC-like protein methyltransferase repurposed for tailoring phosphorylated natural products, and open opportunities to engineer biocatalysts for site-selective aminoglycoside diversification.