Glycosyltransferase Family 38

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

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Glycosyltransferase Family GT38
Clan	GT-B
Mechanisn	inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/GT38.html

Substrate specificities

Members of GT-38 are the bacterial polysialyltransferases (polySTs), which catalyze the addition of sialic acids from the activated sugar donor, CMP-sialic acid (CMP-Neu5Ac), to the nonreducing end of the growing polySia chain [1]. These enzymes build the polymer as a capsular polysaccharide on a specialized poly-β-KDO modified lyso-phosphatidyl glycerol anchor in the membrane of Gram negative bacteria [2]. Bacterial polySia capsules exist in three different flavours: Escherichia coli K1, Neisseria meningitidis serotype B, Moraxella nonliquefaciens, and Mannheimia haemolytica A2 synthesize α-2,8-linked polySia whereas N. meningitidis serotype C produces a α-2,9-linked polymer and E. coli K92 produces polymers with alternating α-2,8 and α-2,9 linkages [3, 4, 5, 6]. In vitro enzyme reactions have shown that the members of GT-38 require two sialic acids for elongation [7, 8, 9], presumably as this mimics the in vivo lipid primer. The enzymes have been used in applications to modify therapeutic proteins and prepare synthetic vaccines [10, 11], where un-natural acceptors like protein N-glycans have been used.

Kinetics and Mechanism

Sialic acid transfer occurs with inversion of configuration (from the β-linked CMP-Neu5Ac donor to the α-2,8-linked polySia), and polyST has been proposed to follow a SN2-like direct displacement mechanism. While H291 could act as a catalytic acid to stabilize the nucleotide phosphate-leaving group.

Catalytic Residues

Residues involved in catalysis have been proposed from site-directed mutagensis and the X-ray crystal structure from the M. hemolytica serotype A2 enzyme [12]. The catalytic base E153 abstracts a proton from the C8′ hydroxyl group of the sialic acid acceptor concerted with the nucleophilic attack on the anomeric C2′ carbon of the CMP-sialic acid donor substrate, thereby generating an α-2,8 glycosidic linkage. The resulting negatively charged CMP leaving group is stabilized by H291 assisted by S339 and T340.

Three-dimensional structures

• PDB ID 5WC6 from GT38 (click images for large versions)
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The structure is a GT-B fold typical of a metal independent glycosyltransferase, it has 2 non-equivalent Rossmann-like folds. The structure was solved from a truncated version of the enzyme, which lacks 20 amino acids from the N-terminal end. There are two protomers in the crystal structure, but biochemical evidence suggests the soluble enzyme exists as a monomer. There is a hinge region between these domains (F227 to N236) which gives some flexibility in the structure. The structure shows an N-terminal tail which is unstructured and is likely to be a linker to the membrane anchor. The structure has a large electropositive groove which accomodates the polySia chain. One of the additional structures obtained for this enzyme is a complex with the synthetic heparin fondaparinux which was a surrogate for the polyanionic polySia. The image shown here is the CDP donor analogue complex which sites on one the Rossmann like domains.

Family Firsts

First general acid/base residue identification

E153, H291

First 3-D structure

PDB 5WC6

References

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