Structural analysis of ASCH domain-containing proteins and their implications for nucleotide processing

ASC-1 homology (ASCH) domain family proteins are believed to play essential roles in RNA metabolism, but detailed structural and functional information is limited. Research has shown that the E. coli enzyme YqfB, which contains an ASCH domain, has amidohydrolase activity, converting N4-acetylcytidine (ac4C) RNA nucleoside into cytidine. Here, we present the crystal structures of EcYqfB both in its unbound state and bound to a substrate. Our analysis reveals how the substrate interacts with the enzyme, offering insights into its catalytic mechanism. In vivo experiments further show that deleting EcYqfB does not change overall ac4C levels across various RNA types, indicating that EcYqfB specifically functions in ac4C nucleoside metabolism. We also determined the structures of two homologous proteins: mouse EOLA1 and the human TRIP4-ASCH domain, highlighting differences in their substrate preferences. These findings offer important insights for future research into the structure and function of the ASCH domain protein family.