Structural Snapshots of α‐1,3‐Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases

Abstract

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double-displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α-1,3-galactosyltransferase (α3GalT) from Bos taurus, which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP-Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front-side substrate-assisted S i-type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.

Structural basis for arginine glycosylation of host substrates by bacterial effector proteins

“…Three reaction mechanisms have been proposed for retaining GTs, namely S N 2, S N i, and orthogonal mechanisms. While the S N 2 mechanism involves a double-displacement reaction requiring a nucleophilic residue to form a covalent glycosyl intermediate 34 , both the S N i and the orthogonal mechanisms involve a single displacement reaction in which the β-phosphate of the nucleotide acts as the catalytic base 35 – 37 . However, the S N i and the orthogonal mechanisms differ in their reaction profiles and the timing of bond formation and bond breakage 37 .…”

“…These differences lead to a dissociative and associative transition state for the S N i and the orthogonal mechanism, respectively 37 . Unlike the S N i mechanism that has been probed extensively, the S N 2 and the orthogonal mechanism have never been demonstrated experimentally 34 – 37 . In our crystal structure, Asn256 SseK1 was located in a possible position for the back-side attack of the C 1 anomeric carbon of UDP-GlcNAc through an S N 2 reaction.…”

Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

“…The reaction mechanism for retaining GTs has been debated for many years, although several recent structures have provided significant insights 22–25 . Similarly to retaining glycosidases that utilize a double-displacement mechanism with a transient covalent enzyme–substrate intermediate 5,9 , retaining GTs were also originally thought to employ a covalent intermediate (Fig.…”

“…1e). Several structural snapshots of enzyme–donor–acceptor Michaelis complexes for retaining GTs have recently appeared (XXYLT 22 , GALNT2 23 , GGTA1 25 , and GpgS 24 ). The position of the nucleophilic acceptor hydroxyl group relative to the anomeric carbon atom, together with snapshots showing progression of the reaction, strongly support the S N i-type mechanism.…”

Comparative Analysis of Radical Adduct Formation (RAF) Products and Antioxidant Pathways between Myricetin-3-O-Galactoside and Myricetin Aglycone

“…Galactosylation is an important biological process in cellular metabolism [1,2] that is catalyzed by certain enzymes, such as bovine β (1,4)-galactosyltransferase [2], Sb3GT1 (UGT78B4) [3], AgUCGalT1 [4], and α-1,3-galactosyltransferase (α3GalT) [5]. Through galactosylation, plant cells can link a galactose residue to the 3- O atom in flavonol.…”