High-resolution crystal structures and STD NMR mapping of human ABO(H) blood group glycosyltransferases in complex with trisaccharide reaction products suggest a molecular basis for product release

Abstract

The human ABO(H) blood group A- and B-synthesizing glycosyltransferases GTA and GTB have been structurally characterized to high resolution in complex with their respective trisaccharide antigen products. These findings are particularly timely and relevant given the dearth of glycosyltransferase structures collected in complex with their saccharide reaction products. GTA and GTB utilize the same acceptor substrates, oligosaccharides terminating with α-l-Fucp-(1→2)-β-d-Galp-OR (where R is a glycolipid or glycoprotein), but use distinct UDP donor sugars, UDP-N-acetylgalactosamine and UDP-galactose, to generate the blood group A (α-l-Fucp-(1→2)[α-d-GalNAcp-(1→3)]-β-d-Galp-OR) and blood group B (α-l-Fucp-(1→2)[α-d-Galp-(1→3)]-β-d-Galp-OR) determinant structures, respectively. Structures of GTA and GTB in complex with their respective trisaccharide products reveal a conflict between the transferred sugar monosaccharide and the β-phosphate of the UDP donor. Mapping of the binding epitopes by saturation transfer difference NMR measurements yielded data consistent with the X-ray structural results. Taken together these data suggest a mechanism of product release where monosaccharide transfer to the H-antigen acceptor induces active site disorder and ejection of the UDP leaving group prior to trisaccharide egress.

Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

“…However, from combined affinity data we suggest that the interplay between UDP, H‐disaccharide, and GTB is similar to the one between UDP‐Gal, H‐disaccharide, and GTB. Enzyme kinetic analysis, recent crystallographic studies of complex formation of GTB with its product, blood group B trisaccharide, as well as theoretical calculations support an ordered kinetic mechanism for GTB, by which the donor substrate binds first. The question of whether a covalent glycosyl–enzyme intermediate is formed is still under debate and cannot be resolved by our experiments.…”

“…Enzyme kinetic analysis, recent crystallographic studies of complex formation of GTB with its product, blood group B trisaccharide, as well as theoretical calculations support an ordered kinetic mechanism for GTB, by which the donor substrate binds first. The question of whether a covalent glycosyl–enzyme intermediate is formed is still under debate and cannot be resolved by our experiments. On the other hand, our study has delivered lifetimes of the ternary GTB, UDP, and H‐disaccharide complex of the order of at least 100 ms.…”

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM‐1 towards Donor Substrates Glucose/Mannose 1‐Phosphate by Using X‐ray Crystallography and Saturation Transfer Difference NMR Spectroscopy

“…This technique has also been used to study enzyme–carbohydrate interactions to elucidate recognition features that can be used for inhibitor design, such as the study of ligand recognition by enzymes involved in mycobacterial cell wall biosynthesis, including uridine diphosphate (UDP) galactopyranose mutase and galactofuranosyltransferases . The same technique has been used to elucidate the binding of human blood group glycosyltransferases to their substrates; a process that is crucial to the biosynthesis of human blood group antigen …”

Insights into Allosteric Control of Human Blood Group A and B Glycosyltransferases from Dynamic NMR

“…Qualitatively, this indicates rather similar bound conformations. On the other hand, a recent crystallographic study came to the conclusion that blood group A/B trisaccharide and UDP cannot be bound at the same time as steric clashes between the β‐phosphate of UDP and the α‐D‐GalNAc/α‐D‐Gal unit of GTA/GTB disorder the active site and prevent UDP from being stably bound. Also, in a mass spectrometry‐based study no binding of B‐trisaccharide to GTB could be detected in the presence of UDP .…”