Structural and biochemical basis for the formation of α-(1→3)-linked mannose residue in O-glycans of Saccharomyces cerevisiae.

Abstract

The biosynthesis of O-mannose-type glycans in the budding yeast Saccharomyces cerevisiae requires the sequential action of multiple mannosyltransferases. However, the molecular mechanism governing α-(1→3)-linkage formation remains poorly understood. Mnt2p, a member of glycosyltransferase family 71 (GT71), catalyzes the transfer of the fourth and fifth mannose residues of O-glycans via α-(1→3)-linkages. Here, we report the crystal structure of Mnt2p at 2.40 Å resolution, representing the first experimentally determined structure of a GT71 family enzyme. The structure revealed an N-terminal domain adopting a canonical GT-A fold harboring a DXD motif that coordinates a Mn2+ ion and a novel C-terminal domain unique to the GT71 family. The active site is formed at the interface between these two domains, and GDP-mannose binds to a cleft adjacent to the catalytic metal ion. Using recombinant Mnt2p expressed in Escherichia coli, we biochemically demonstrated its α-(1→3)-mannosyltransferase activity and determined its substrate specificity toward α-mannobiose acceptors. Docking and molecular dynamics simulations revealed that the C-terminal domain forms the acceptor substrate-binding site, providing the structural basis for α-(1→3)-linkage selectivity, distinct from the linkage specificity of α-(1→2)-mannosyltransferases. These findings establish the structural foundation for understanding the differentiation of α-(1→2) and α-(1→3) mannosyl linkages in yeast O-glycan biosynthesis.