Structural basis for the carboxylation and epoxidation of human gamma-glutamyl carboxylase

Gamma-glutamyl carboxylase (GGCX) is the sole enzyme responsible for gamma carboxylation of glutamate in a vitamin K-dependent manner. This process is crucial for blood coagulation, bone metabolism, vascular calcification, and other biological processes because gamma carboxylation is essential for the maturation of clotting factors, anticoagulation factors, and some coagulation-unrelated factors. Despite these essential roles, the catalytic mechanism of GGCX remains incompletely understood. Here, we present the cryo-EM structures of human GGCX complexed with five typical substrates, including two clotting factors and three coagulation-unrelated factors. These structures not only elucidate the recognition mechanism for the propeptide but also reveal three distinct modes for substrate loading. Among them, the GGCX-MGP complex structure reveals a specific mode to load a substrate with an active glutamate residue at the N-terminus of the propeptide. Moreover, these structural observations are supported by our in vitro carboxylation and epoxidation assays.