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	retaining/inverting
Active site residues	His188 is the catalytic base in the Campylobacter jejuni GT-42 known as Cst-II
CAZy DB link
http://www.cazy.org/fam/GT42.html

Substrate 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](Gilbert 2008). The CjGT-42 known as Cst-II was found as part of the LOS biosynthesis operon [1](Gilbert et al. 2000), 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 [1](Gilbert et al. 2002). 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 [1](Koga et al. 2005) (van Belkum et al. 2001). 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 [1](Fox et al. 2006). 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 [1](Schweda et al. 2007).

This is an example of how to make references to a journal article [1]. (See the References section below). Multiple references can go in the same place like this [1, 2]. You can even cite books using just the ISBN [3]. References that are not in PubMed can be typed in by hand [4].

Kinetics and Mechanism

Content is to be added here. Normal 0 false false false MicrosoftInternetExplorer4 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. His188 appearsed to be the catalytic base, which is somewhat unusual since this role is generally performed by Glu or Asp residues. We collaborated with Dr; Lawrence McIntosh (Chan et al 2009, Biochemistry 48: 11220-30 attached) to directly measure its pKa by NMR and show that the pKa measured agrees with that deduced from the pH dependence of this reaction. This required the generation of an active monomeric form of the tetrameric enzyme. We were also able to perform chemical rescue of the H188A mutant with nucleophilic anions adding weight to the assignment of the function, and further supporting the SN2-like inverting mechanism.

Catalytic Residues

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Three-dimensional structures

The CjGT-42 enzymes Cst-I and Cst-II were the first sialyltransferases whose structure was determined by X-ray crystallography [1](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 (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 (Fig. 8) 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 (Fig. 9)

Family Firsts

First stereochemistry determination

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

First catalytic nucleophile identification

Cite some reference here, with a short (1-2 sentence) explanation [4].

First general acid/base residue identification

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

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [3].

References

1. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n |
2. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 |
3. Robert V. Stick and Spencer J. Williams. (2009) Carbohydrates. Elsevier Science.

4. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006