X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily

Abstract

N-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N-glycans and plays a critical role in mammalian development and possibly all metazoans. We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn 2+ at 1.5 and 1.8 A Ê resolution, respectively. The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining b-glycosidases. The structuring of a 13 residue loop, resulting from UDPGlcNAc/Mn 2+ binding, provides an explanation for the ordered sequential`Bi Bi' kinetics shown by GnT I. Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine b-1,4-galactosyltransferase 1 and Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase. The low sequence identity, conserved fold and related functional features shown by this domain de®ne a superfamily whose members probably share a common ancestor. Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.

Structure of Kre2p/Mnt1p

“…The ribose is bound in the 2Ј-endo conformation, similar to that seen in ␣1,4-N-acetylhexosaminyltransferase (EXTL2) (18), GnT I (18,34) and MobA (42). Interestingly, in other retaining glycosyltransferases such as LgtC (20), ␣1,3GalT (17,19) and glycogenin (21), the ribose is bound in the 3Ј-endo conformation.…”

“…Refs. 17,20,34)). These conformational changes sequester the active site from the solvent and are also suggested to be involved in product release (25).…”

Crystal Structure of β1,3-Glucuronyltransferase I in Complex with Active Donor Substrate UDP-GlcUA

“…The catalytic region of GlcAT-I shows a great deal of similarity to other inverting glycosyltransferases despite little to no sequence identity. The enzymes N-acetylglucosaminyltransferase I from rabbit (GnT1) (21) and SpsA from Bacillus subtilis (19) both share a great deal of structural similarity not only with the nucleotide binding subdomain of GlcAT-I but the overall fold as well. ␤1,4-Galactosyltransferase from bovine (␤4GalT1) (22-24) also shows some similarities in overall fold to GlcAT-I.…”

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

“…These enzymes have low sequence homology, and because there was no structural information on them until recently, they have been classified into dozens of different families (1) (http:͞͞afmb.cnrs-mrs.fr͞ϳcazy͞CA ZY͞in-dex.html). In the past few years, 11 different UDP͞TDP-GTase structures from 10 different families have been reported (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), but all belong to only two different structural superfamilies, GT-A and GT-B (16). Eight of the eleven crystal structures obtained to date belong to the GT-A superfamily, which includes most of the GTases found in the Golgi apparatus and the endoplasmic reticulum.…”