Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

Abstract

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.

Recent structural and mechanistic insights into protein O-GalNAc glycosylation

“…These included the first Michaelis complex structure of GalNAc-T2 containing UDP-5SGalNAc with the mEA2 peptide ( Figure 2a), as well as a product complex containing UDP and the glycosylated EA2 peptide (Figure 2b). Furthermore, a pre-Michaelis complex was also trapped, in which the oxygen atom of the hydroxy group of the acceptor Thr is not properly located to attack the anomeric carbon of the GalNAc moiety ( Figure 2b) [24].…”

“…Hydrogen bonds (green dashed lines) are only depicted for residues interacting with the sugar moiety and α-and β-phosphate for illustration purposes. Protein-Mn 2 + coordination is shown as dotted olive lines [24]. (b) Overlay of the ternary substrate complexes and product complex.…”

A Native Ternary Complex Trapped in a Crystal Reveals the Catalytic Mechanism of a Retaining Glycosyltransferase

“…[1,10,11,12] The concept of a general internal nucleophilic substitution mechanism S N i, which is neither simply S N 1 nor S N 2, was first invoked to explain unusual stereochemical outcomes of simple alkyl halides, and then proposed for glycosyl transfer mechanism, in the absence of any enzyme, to explain the retention of anomeric stereochemistry in the solvolysis of α-glucosyl fluoride by mixtures of ethanol and trifluorethanol. [10] In this mechanism leaving group departure and nucleophilic attack occur on the same face of the sugar [13] , involving either a short-lived oxocarbenium ion intermediate (S N i-like) [6,9,14,15,16] or an oxocarbenium ion transition state (S N i).…”

“…[16] The LgtC Michaelis complex was modeled based on a crystal structure containing two substrate analogues, UDP 2-deoxy-2'-F-Gal and 4-deoxylactose. The attacking hydroxyl group of lactose has the oxygen atom O4 at a distance of 3.1 Å from the anomeric carbon C' of the donor galactose moiety, and at 2.7 Å from the glycosidic oxygen atom.…”

“…Extensive theoretical studies using QM/MM calculation simulations supported the occurrence of the S N i 'internal return' mechanism in OtsA, LgtC and GalNAc-T2. [15,16,17,21] We have employed molecular dynamics simulations combined with QM(DFT)/MM calculations to further understand the reaction mechanism followed by GpgS (see SI Section for details; Figure 4 and S9). The sugar transfer was modeled using the reaction…”