Difference between revisions of "Glycosyltransferase Family 42"

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

Glycosyltransferase Family GT42
Clan	GT-x
Mechanism	inverting, donor is CMP-beta-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 a2,8-a2,3 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 a-2,3 and a-2,8 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 a-2,3-sialyltransferase Lic3A (Hood et al. 2001), followed by a second a-2,3/2,8-bi-functional version known as Lic3B [6]. 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 [7].

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 [8]. 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 [8]. 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 from strain OH4383 appears to be the catalytic base.

Three-dimensional structures

The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography [9][10], 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. 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.

  Normal  0      false  false  false                     MicrosoftInternetExplorer4       Glycosyltransferase structures often have a “lid” domain, which is a mobile loop that undergoes a conformational change upon binding a substrate (Lairson et al. 2008).  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 (Chiu et al. 2007).  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 (Chiu et al. 2007).

Family Firsts

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation

First 3-D structure

The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography (Chiu et al. 2004), (Chiu et al. 2007), 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)
top view
side view

References

All Medline abstracts: PubMed

@@ Line 15: / Line 15: @@
 |-
 |'''Mechanism'''
-|retaining/inverting
+|inverting, donor is CMP-beta-Neu5Ac
 |-
 |'''Active site residues'''
-|His188 is the catalytic base in the Campylobacter jejuni GT-42 known as Cst-II
+|His188 is the catalytic base in the ''Campylobacter jejuni'' GT-42 known as Cst-II
+|His201 is the catlytic base in the''Campylobacter jejuni''GT42 known as Cst-I
 |-
 |{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
@@ Line 28: / Line 29: @@
-== Substrate specificities ==
+== 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 a2,8-a2,3 linked di-sialic acid moiety <cite>Gilbert2008</cite>.  The CjGT-42 known as Cst-II was found as part of the LOS biosynthesis operon <cite>Gilbert2000</cite>, which facilitated the correlation of gene content to LOS structure.  Based on the LOS structures which contained both a-2,3 and a-2,8 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 <cite>Gilbert2002</cite>.  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'' <cite>Koga2005</cite> <cite>vanBelkum2001</cite>.  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 a-2,3-sialyltransferase Lic3A (Hood et al. 2001), followed by a second a-2,3/2,8-bi-functional version known as Lic3B <cite>Fox2006</cite>.  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'' <cite>Schweda2007</cite>.
@@ Line 42: / Line 43: @@
 == Three-dimensional structures ==
-The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography <cite>chiu2004</cite><cite>chiu2007</cite>, 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 (Fig. 10).  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.
+The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography <cite>chiu2004</cite><cite>chiu2007</cite>, 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.  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.
+   Normal  0      false  false  false                     MicrosoftInternetExplorer4       Glycosyltransferase structures often have a “lid” domain, which is a mobile loop that undergoes a conformational change upon binding a substrate (Lairson et al. 2008).  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 (Chiu et al. 2007).  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 (Chiu et al. 2007).