The Influence of an Intramolecular Hydrogen Bond in Differential Recognition of Inhibitory Acceptor Analogs by Human ABO(H) Blood Group A and B Glycosyltransferases

Abstract

Human ABO(H) blood group glycosyltransferases GTA and GTB catalyze the final monosaccharide addition in the biosynthesis of the human A and B blood group antigens. GTA and GTB utilize a common acceptor, the H antigen disaccharide ␣-L-Fucp-(132)-␤-D-Galp-OR, but different donors, where GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal. GTA and GTB are two of the most homologous enzymes known to transfer different donors and differ in only 4 amino acid residues, but one in particular (Leu/Met-266) has been shown to dominate the selection between donor sugars. The structures of the A and B glycosyltransferases have been determined to high resolution in complex with two inhibitory acceptor analogs ␣-L-Fucp-(132)-␤-D-(3-deoxy)-Galp-OR and ␣-L-Fucp-(132)-␤-D-(3-amino)-Galp-OR, in which the 3-hydroxyl moiety of theGal ring has been replaced by hydrogen or an amino group, respectively. Remarkably, although the 3-deoxy inhibitor occupies the same conformation and position observed for the native H antigen in GTA and GTB, the 3-amino analog is recognized differently by the two enzymes. The 3-amino substitution introduces a novel intramolecular hydrogen bond between O 2 on Fuc and N3 on Gal, which alters the minimum-energy conformation of the inhibitor. In the absence of UDP, the 3-amino analog can be accommodated by either GTA or GTB with the L-Fuc residue partially occupying the vacant UDP binding site. However, in the presence of UDP, the analog is forced to abandon the intramolecular hydrogen bond, and the L-Fuc residue is shifted to a less ordered conformation. Further, the residue Leu/Met-266 that was thought important only in distinguishing between donor substrates is observed to interact differently with the 3-amino acceptor analog in GTA and GTB. These observations explain why the 3-deoxy analog acts as a competitive inhibitor of the glycosyltransferase reaction, whereas the 3-amino analog displays complex modes of inhibition.

Differential Recognition of the Type I and II H Antigen Acceptors by the Human ABO(H) Blood Group A and B Glycosyltransferases

“…Initial designs of inhibitors of GTA and GTB were made in the absence of any structural information concerning GTs and consisted of chemical modification of hydroxyl groups on the central galactose residue of HA-octyl (12,13). These studies have provided some of the few GT⅐inhibitor complexes available (11). In the present study, both enzymes have been shown to form an overwhelming number of interactions with the Gal residue such that its location in the active site is reproduced regardless of the other substituted saccharides.…”

“…This pattern has also been seen in inhibitory analogs of the H antigen, where the galactose residue analog dominates binding, and modification of specific points on the galactose residue can lead to effective inhibition (11)(12)(13).…”

ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes

“…Crystallization-All proteins were crystallized using conditions different from those reported previously (10,19,33,35,38,39). Whereas the first crystals of GTB were grown from relatively low protein concentrations (ϳ8 -15 mg/ml) and as a mercury derivative, the crystals in this paper were initially generated from higher protein concentrations (ϳ60 -75 mg/ml).…”

“…Elucidation of the details of substrate recognition would allow the development of new inhibitors for the treatment of microbial diseases (5), genetic ailments such as diabetes (6), and cancer (7). The generation of inhibitors of the blood group A and B synthesizing glycosyltransferases GTA 2 and GTB have been reported (8,9), including an inhibitor-bound structure (10).…”

Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase

“…The glycosyltransferase GTA transfers the N-acetylgalactosaminyl monosaccharide residue from UDP to Gal O3 of the H antigen [minimal epitope Fuc-(1!2)Gal] with retention of the axial stereochemistry to generate the human blood group A antigen and is a model enzyme for studying the retaining glycosyltransfer mechanism (Alfaro et al, 2008;Lee et al, 2005;Letts et al, 2007;Nguyen et al, 2003;Patenaude et al, 2002;Persson et al, 2007;Schuman et al, 2007Schuman et al, , 2010Seto et al, 1999). Evaluation of hydrogenbonding partners and amino-acid protonation states is critical for understanding the catalytic mechanisms of GTA and indeed of all retaining glycosyltransferases.…”