Fig. 6: Proposed catalytic mechanism of TxgGalNAc-T3.
a Structure of TxgGalNAc-T3 (catalytic domain in lavender and lectin domain in light grey) reveals unique characteristics, including a second Mn2+N (aquamarine), a charged residue Glu332 in the active site close to the acceptor Thr, a flexible loop II (purple) that interacts with the substrate peptide mainchain, and an extended C-terminal tail (black). b Human GalNAc-T12 (catalytic domain in salmon and lectin domain in light blue) is missing these features but contains a GalNAc binding pocket in the lectin domain, which has not been shown to be present in the lectin domain of TxgGalNAc-T3. c SNi retaining catalytic mechanism of a human GalNAc-T (hGalNAc-T2). Here, the acceptor Thr approaches the anomeric carbon in a front-face reaction, resulting in the formation of an oxocarbenium ion. The β phosphate on the UDP leaving group extracts a proton from Thr as a bond forms between the acceptor and GalNAc with retention of configuration. d Proposed double-displacement catalytic mechanism of TxgGalNAc-T3. Glu332 could act as a nucleophile and initiate catalysis by approaching the C1 carbon on GalNAc for a nucleophilic attack, resulting in inversion of configuration to a β−linked GalNAc. The acceptor Thr then forms a bond with the anomeric carbon of GalNAc in an SN2 type nucleophilic reaction to displace Glu332 and retain the α stereochemistry on GalNAc in the final product.
