Glycosyltransferase Family 42

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• Author: ^^^Warren Wakarchuk^^^
• Responsible Curator: ^^^Warren Wakarchuk^^^

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Glycosyltransferase Family GT42
Clan	GT-x
Mechanism	CMP-NeuAc α-2,3-sialyltransferase (EC 2.4.99.-)which is inverting, donor is CMP-b-Neu5Ac
Active site residues	His188 is the catalytic base in the Campylobacter jejuni GT-42 known as Cst-II His201 is the catlytic base in theCampylobacter jejuniGT42 known as Cst-I
CAZy DB link
http://www.cazy.org/fam/GT42.html

Acceptor specificities

The enzymes in GT-42 were originally examined from isolates of C. jejuni that express a number of ganglioside mimics, some of which have an α2,8-α2,3 linked di-sialic acid moiety [1]. The CjGT-42 known as Cst-II was found as part of the LOS biosynthesis operon [2], which facilitated the correlation of gene content to LOS structure. Based on the LOS structures which contained both α2,3 and α2,8 linked sialic acid it was predicted that there should be two sialyltransferases, but the surprising finding was that a single CjGT-42 enzyme, Cst-II, was making both linkages [3]. This bi-functional enzyme has been shown to be a determinant in the development of post-infectious neuropathies (Guillain-Barré syndrome), due to an anti-ganglioside antibody response in some patients who were infected with C. jejuni [4, 5]. Some species of Campylobacter express a second GT-42 enzyme known as Cst-I, which was in fact cloned first, but it has been shown not to be involved in LOS biosynthesis on the basis of gene knockouts in strains with both cst genes (Michel Gilbert, personal communication). Cst-I contains an extended C-terminal domain of unknown function, and the target acceptor of this second CjGT-42 is not known at present. The GT-42 family also contains members from H. influenzae and P. multocida, in which the GT-42 enzymes are one of two or three resident sialyltransferases. In H. influenzae the first GT-42 enzyme to be described was the α2,3-sialyltransferase Lic3A [6], followed by a second a2,3/2,8-bi-functional version known as Lic3B [7]. In H. influenzae the in vivo acceptor for Lic3A/B is the simple disaccharide lactose in contrast to the more complex ganglioside mimics seen in C. jejuni [8]. At present there are uncharacterized members of GT42 from Helicobacter acinonychis str. Sheeba, H. mustelae which were identified through genome sequencing, but which have not been linked to specific sialylated structures.

Kinetics and Mechanism

Detailed kinetic studies have revealed that this enzyme follows an unusual steady state iso ordered bi bi kinetic mechanism for carrying out sialyl transfer with inversion [9]. His188 appears to be the catalytic base, which is somewhat unusual since this role is generally performed by Glu or Asp residues. Direct measurement of its pKa by NMR showed the pKa agrees with that deduced from the pH dependence of this reaction. This required the generation of an active monomeric form of the tetrameric enzyme [9]. Chemical rescue of the H188A mutant was performed with nucleophilic anions adding weight to the assignment of the function, and further supporting the SN2-like inverting mechanism.

Catalytic Residues

His188 in CjGT-42 Cst-II_OH4383 appears to be the catalytic base. The amino acids Asn51 and Leu54 had the most effect on the bi-functional activity of Cst-II, but only Asn51 was shown to be critical for the α2,8-sialyltransferase activity in this variant of Cst-II. There are members of GT42 which have an Asn at an equivalent sequence position that are not bi-functional, but they have much lower overall sequence homolgy with Cst-II_OH4384.

Three-dimensional structures

The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography [10, 11], and they share the same basic structure. GT-42 enzymes have a modified GT-A type fold characterized by a single α/b Rossmann fold nucleotide binding domain, and a flexible lid domain composed of a coil and two helices. Unlike the typical GT-A fold, CjGT-42 enzymes lack the conserved DXD motif and are metal-ion independent. The enzyme structure revealed a tetramer in which each monomer carries an independent active site, and examination of the active site residues suggested a conserved histidine, His188 in Cst-II, as the catalytic base.

Glycosyltransferase structures often have a “lid” domain, which is a mobile loop that undergoes a conformational change upon binding a substrate [12]. The lid domain in Cst-I and Cst-II contains residues which interact with the donor and/or acceptor. A common feature of transferase substrate binding is the role of aromatic residues stacking onto the base of the sugar-nucleotide. In Cst-I the aromatic residue is Tyr171 and in Cst-II it is Tyr156, both of which are conserved in GT-42 and occur in the lid domain. There is no structure of a ternary complex for GT-42 so it is not possible to know all of the interactions that are involved in substrate binding, but there are donor complexes with the incompetent donor CMP-3-fluoro-Neu5Ac and acceptor models have been examined to predict interactions [11]. In Cst-I, Phe190 appears to prevent sialylated acceptors from binding in the active site. In Cst-II, this Phe residue is replaced by Ala175, which opens up a pocket for the carboxyl group of a modelled sialyl-lactose acceptor [11].

Family Firsts

The CjGT-42 enzyme Cst-II from strain OH4384, was the demonstrated to be first sialyltransferase which could make both a2,3 and a2,8 linkages [3]. This bi-functional activity is unique to GT-42.

The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography [10, 11], and they share the same basic structure. GT-42 enzymes have a modified GT-A type fold characterized by a single a/b Rossmann fold nucleotide binding domain, and a flexible lid domain composed of a coil and two helices.

• PDB ID 1RO8 from GT42 (click images for large versions)
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References

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1. Irving Nachamkin, Christine M. Szymanski, and Martin J. Blaser. (2008) Campylobacter. Amer Society for Microbiology.

   Book chapter: Gilbert, M., Parker, C.T., and Moran, A. P. Campylobacter jejuni Lipooligosaccharides: Structure and Function. In Campylobacter 3rd Edition, editors Irving Nachamkin, Christine M. Szymanski, Martin J. Blaser, ASM press, Washington D.C. pp 438-504, 2008