Structure and function of metal-independent CAZy family 6 glycosyltransferase from Bacteroides ovatus

Abstract

Glycosyltransferases (GTs) are an enzyme superfamily responsible for the synthesis of glyconjugates by transferring the sugar moiety from an active donor to a specific acceptor, usually a protein or a polysaccharide. The glyconjugates, such as glycolipids and glycoproteins, play vital roles in physical maintenance of tissue structures, immune recognition and other biological activities. Understanding their catalytic mechanisms is therefore critical. There are two kinds of reaction defined based on the stereochemistry of the product carbohydrate moiety compared to the donor: retaining and inverting. Whilst the mechanism of inverting GTs is well-known, the catalytic process of retaining GTs is as yet unclear.Glycosyltransferase family 6 (GT6), according to the Carbohydrate Active Enzyme (CAZy) database, is a retaining GT family which catalyses the transfer of α-galactose (α-Gal) or α-N-acetyl-galactosamine (α-GalNAc) to the 3-OH group of a β-linked Gal or GalNAc in an acceptor substrate. Most GT6s from vertebrates require a metal ion for their activity. The metal ion-dependence is linked to the AspXaaAsp (DXD) motif which is conserved among these enzymes. However, analysing sequences of GT6s from bacteria showed that the DXD motif was substituted by an AsnXaaAsn (NXN) sequence. One of two CAZy family 6 glycosyltransferases, BoGT6a from Bacteroides ovatus, which catalyses the transfer of GalNAc from UDP-GalNAc to the saccharide acceptor and UDP-GalNAc hydrolysis, was kinetically and structurally studied. This enzyme is fully active in the absence of metal ions. The structure of BoGT6a is strikingly similar to its mammalian homologues such as GTA, GTB and α-1,3-galactosyltransferase, but it has a shorter N-terminal region and a NXN motif instead of a DXD motif. This suggests that the substitution of the DXD motif with the NXN may affect the catalytic mechanism of the enzyme.The structure of the enzyme in complex with its acceptor molecule 2’-fucosyllactose was obtained at 3.0 Å. Comparison of the X-ray crystallographic structures of BoGT6a in its native and acceptor bound forms demonstrated the conformational changes of the enzyme associated with acceptor binding. It also elucidated the impact of acceptor binding on enzyme conformation and the structural relationship between the enzyme and its homologues.Structural snapshots of the BoGT6a Glu192Gln (E192Q) mutant processing its donor UDP-GalNAc were also obtained. The interactions between the enzyme and the donor provide an insight into the mechanistic role of the NXN motif and nearby amino acid residues in BoGT6a’s metal-independent activity. Moreover, the high flexibility of the enzyme conformation when it interacts with the ligands provides a general picture of how the enzyme processes UDP-GalNAc.Together, these structures illustrate how a significant divergence in catalytic properties can be accommodated by minor structural adjustments, and propose a role for the NXN motif, which replaces the DXD motif in the metal independent glycosyltransferases.

Date of Award	31 Aug 2014
Original language	English
Awarding Institution	University of Bath
Sponsors	Biotechnology Center of Ho Chi Minh City
Supervisor	Ravi Acharya (Supervisor)