Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

Abstract

Glycans linked to proteins and lipids play key roles in biology; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division. The fact that glycans are not copied from genomic templates suggests that fidelity is provided by the catalytic templates of glycosyltransferases that accurately add sugars to specific locations on growing oligosaccharides. To form new glycosidic bonds, glycosyltransferases bind acceptor substrates and orient a specific hydroxyl group, frequently one of many, for attack of the donor sugar anomeric carbon. Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. The varied approaches for acceptor binding site assembly suggest an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures observed in biology.

Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases

“…We find that natural perturbations in the catalytic base residue, an important distinction between the inverting and retaining mechanisms, correlates well with these multiple emergences across the tree. The residue that acts as a catalytic base for inverting GTs (aspartate within the xED motif, xED-Asp) is variable across the retaining families consistent with its lack of role in the retaining S N i mechanism (6). In the inverting families, the xED-Asp is nearly always conserved and appropriately positioned to function as a catalytic base (Fig.…”

“…In the inverting families, the xED-Asp is nearly always conserved and appropriately positioned to function as a catalytic base (Fig. 3A), though some exceptions have been noted (6, 25). Out of the five clades grouping inverting and retaining families, inverting families in three of these clades do not conserve the xED-Asp (GT2-DPs, GT2-LpsRelated and GT43).…”

“…Inverting mechanisms require an in-line attack and direct displacement by the nucleophile relative to the departing nucleotide diphosphate of the sugar donor and a conserved placement of the xED-Asp carboxyl group as catalytic base at the beginning of the αF helix. In contrast, retaining enzymes generally alter the angle of nucleophilic attack by the acceptor, use a donor phosphate oxygen as catalytic base, and employ a dissociative mechanism for sugar transfer (6). The fundamental differences in these catalytic strategies would suggest an early divergence of enzymes employing these respective mechanisms.…”

“…While the sequence basis for inverting and retaining mechanisms is not well understood, most inverting GT-As have a conserved Asp or Glu within a xED motif that serves as the catalytic base to deprotonate the incoming nucleophile of the acceptor, and initiate nucleophilic attack with direct displacement of the phosphate leaving group (7, 8). Retaining GT-As bind the sugar donor similarly to the inverting enzymes, but shift the position of the acceptor nucleophile to attack the anomeric carbon from an obtuse angle using a phosphate oxygen of the sugar donor as the catalytic base and employ a dissociative mechanism that retains the anomeric linkage for the resulting glycosidic product (6). Such mechanistic diversity of GTs is further illustrated by recent crystal structures of GTs bound to acceptor and donor substrates which show that different acceptors are accommodated in the active site through variable loop regions emanating from the catalytic core (6).…”

“…These families can be broadly classified into two categories based on their mechanism of action and the anomeric configuration of the glycosidic product relative to the sugar donor, namely, inverting or retaining. Inverting GTs generally employ an S N 2 single displacement reaction mechanism that results in inversion of anomeric configuration for the product, whereas retaining GTs generally employ a dissociative S N i-type mechanism that retains the anomeric configuration of the product (6). While the sequence basis for inverting and retaining mechanisms is not well understood, most inverting GT-As have a conserved Asp or Glu within a xED motif that serves as the catalytic base to deprotonate the incoming nucleophile of the acceptor, and initiate nucleophilic attack with direct displacement of the phosphate leaving group (7, 8).…”