Human N-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAc beta 1,2-branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAc-Man(3)GlcNAc(2)-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GTA Rossmann-like fold that employs conserved divalent cationdependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-alpha 1,6-mono-saccharide acceptor and a distal exosite pocket that binds the GlcNAc-beta 1,2Man-alpha 1,3Man beta-substrate recognition arm. Recognition arm interactions are similar to the enzyme-substrate interactions for Golgi a-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.