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Fold Variations

Reprinted from original article with permission from ASBMB

Core 2 O-glycans are important ligands for selectin-mediated lymphocyte homing and leukocyte rolling. X-ray Crystal Structure of Leukocyte Type Core 2 1,6-N-Acetylglucosaminyltransferase (C2GnT-L) is a metal ion-independent glycosyltransferase that catalyses the synthesis of core 2 branched O-glycans in heart, brain, placenta, spleen, peripheral leukocytes, lymph node and bone marrow. Pak et al. have determined the X-ray crystal structure of C2GnT-L in the absence and presence of its acceptor substrate, Gal1-3GalNAc. Their findings published in the Journal of Biological Chemistry shed new light on the molecular basis of the enzyme's catalytic mechanism.

Previous studies have shown that glycosyltransferases (GT) have either a GT-A or GT-B fold. The GT-A fold consists of two tightly associated α domains and is present in metal ion-dependent GTs. The GT-B fold is characterized by two similar, but less tightly associated Rossman-α domains and is found in most ion-independent GTs. C2GnT-L is an exception to the rule, as despite being a metal ion-independent enzyme, it possesses a GT-A fold.  

Pak et al. show that there are no significant structural changes upon binding of the acceptor substrate. Gal1-3GalNAc binding is mediated by several hydrogen bonds, and occurs at a solvent exposed cleft where residues known to be crucial for acceptor substrate binding are found. Five of the six amino acids that are directly involved in Gal1-3GalNAc binding are conserved between C2GnT-L and two other GT-14 family members, C2GnT-M and C2GnT-T.

Interestingly, unlike most GT-A fold transferases, C2GnT-L does not require the donor substrate to bind in order for the acceptor binding site to form, it is essentially 'preformed'. What's more, C2GnT-L's fold does not contain the characteristic metal ion-binding DXD motif. Instead, Arg-378 and Lys-401, highly conserved among GT-14 family members, electrostatically stabilize the nucleoside diphosphate leaving group, thus serving the role of a metal ion. The chemical environments surrounding the leaving group in C2GnT-L and other GT-B fold ion-independent GTs are remarkably similar, suggesting that the catalytic mechanisms of some GT-A and GT-B transferases have converged.

The unexpected fold variations of C2GnT-L and CstII, another GT whose structure has also been determined recently, challenge the classification of GTs into two superfamilies on the basis of their folds. This study in particular, suggests that despite the fact that not all GT-A and GT-B folds posses the characteristic features we have grown to expect, similar catalytic mechanisms are found across fold types.

Author: Monica Hoyos Flight