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

Abstract

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 -acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn-UDP donor analog complex and as a GlcNAcManGlcNAc-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent 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-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate "recognition arm." Recognition arm interactions are similar to the enzyme-substrate interactions for Golgi α-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.

Structure and mechanism of cancer-associated N-acetylglucosaminyltransferase-V

“…As both of the two middle domains take Rossmann folds, GnT-V is classified into the GT-B fold. This is consistent with the previous prediction by homology modeling 26 and is in marked contrast to other mammalian GlcNAc transferases such as GnT-I (GT13) 27 , GnT-II (GT16) 28 , POMGnT1 29 , and C2GnT-L 30 , which are classified into GT-A folds (Supplementary Figure 1 ). The GT-A fold contains a single Rossmann-fold and usually has the DXD motif, which is critical for binding a divalent cation.…”

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

“…4b). The presence of exosites for intimate binding to N-glycans was also observed in distant glycosyltransferases (GTs) and glycosyl hydrolases such as MGAT2 and MAN2A1, respectively 23 . Our structure also exhibits two striking conformational changes relative to the apo form (RMSD of 0.56 Å on 436 equivalent Cαs; Fig.…”

3D Structure and Function of Glycosyltransferases Involved in N-glycan Maturation

“…Crystal structures of the human GnT-II catalytic domain UO 2 derivative, Mn 2+ -UDP complex, and acceptor (GlcNAcMan 3 GlcNAc 2 -Asn) complex were recently determined at 2.0, 1.6, and 2.8 Å resolutions, respectively [28]. The overall fold of human GnT-II consists of an eight-stranded twisted β-sheet with 12 α-helical segments and forms GT-A fold such as GnT-I ( Figure 3a).…”