Abstact

Sulfotransferases (SULTs) can transfer sulfonate group from a donor molecule, typically 3'-phosphoadenosine-5'-phosphosulfate (PAPS), to a variety of acceptor molecules including hormones, drugs, and xenobiotics, thus play key roles in animal xenobiotic metabolisms and hormone regulations. Here, the first protein crystal structure of a chicken SULT1B1 BtSULT1B1 was obtained and its catalytic mechanism and substrate binding mode was elucidated by analyzing its structures in complex with substrates and donors like PAP, PAPS, pNP, pNPS and 2-Bromophenol. Notably, the gating loop of the substrate-binding pocket of BtSULT1B1 exhibits an enlarged cavity compared to homologous SULT structures from Human and Mouse, facilitating the acceptance of bulky substrates/products. Through conservative amino acid analysis and site-directed mutagenesis, a variant with 3.6-fold enhanced activity, A44S was obtained, and its mechanism was further illustrated by molecular dynamics simulations. The findings of the chicken SULT1B1 in this study expand the knowledge of substrate binding of sulfotransferases, and offer a rational approach for engineering enzymes with improved activities for specific applications, provide molecular basis for the design of in vitro sulfation platform and drug development.