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

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Family Firsts

First stereochemistry determination

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First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

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References

1. Cho JW and Troy FA 2nd. (1994). Polysialic acid engineering: synthesis of polysialylated neoglycosphingolipids by using the polysialyltransferase from neuroinvasive Escherichia coli K1. Proc Natl Acad Sci U S A. 1994;91(24):11427-31. DOI:10.1073/pnas.91.24.11427 |
2. Willis LM, Stupak J, Richards MR, Lowary TL, Li J, and Whitfield C. (2013). Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens. Proc Natl Acad Sci U S A. 2013;110(19):7868-73. DOI:10.1073/pnas.1222317110 |
2. Willis LM, Stupak J, Richards MR, Lowary TL, Li J, and Whitfield C. (2013). Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens. Proc Natl Acad Sci U S A. 2013;110(19):7868-73. DOI:10.1073/pnas.1222317110 |
3. Jennings HJ, Bhattacharjee AK, Bundle DR, Kenny CP, Martin A, and Smith IC. (1977). Strucutres of the capsular polysaccharides of Neisseria meningitidis as determined by 13C-nuclear magnetic resonance spectroscopy. J Infect Dis. 1977;136 Suppl:S78-83. DOI:10.1093/infdis/136.supplement.s78 |
4. Puente-Polledo L, Reglero A, González-Clemente C, Rodríguez-Aparicio LB, and Ferrero MA. (1998). Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2. Glycoconj J. 1998;15(9):855-61. DOI:10.1023/a:1006902931032 |
5. Devi SJ, Schneerson R, Egan W, Vann WF, Robbins JB, and Shiloach J. (1991). Identity between polysaccharide antigens of Moraxella nonliquefaciens, group B Neisseria meningitidis, and Escherichia coli K1 (non-O acetylated). Infect Immun. 1991;59(2):732-6. DOI:10.1128/iai.59.2.732-736.1991 |
6. Glode MP, Robbins JB, Liu TY, Gotschlich EC, Orskov I, and Orskov F. (1977). Cross-antigenicity and immunogenicity between capsular polysaccharides of group C Neisseria meningitidis and of Escherichia coli K92. J Infect Dis. 1977;135(1):94-104. DOI:10.1093/infdis/135.1.94 |
7. Willis LM, Gilbert M, Karwaski MF, Blanchard MC, and Wakarchuk WW. (2008). Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. Glycobiology. 2008;18(2):177-86. DOI:10.1093/glycob/cwm126 |
8. Peterson DC, Arakere G, Vionnet J, McCarthy PC, and Vann WF. (2011). Characterization and acceptor preference of a soluble meningococcal group C polysialyltransferase. J Bacteriol. 2011;193(7):1576-82. DOI:10.1128/JB.00924-10 |
9. Lindhout T, Bainbridge CR, Costain WJ, Gilbert M, and Wakarchuk WW. (2013). Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases. PLoS One. 2013;8(7):e69888. DOI:10.1371/journal.pone.0069888 |
10. Lindhout T, Iqbal U, Willis LM, Reid AN, Li J, Liu X, Moreno M, and Wakarchuk WW. (2011). Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. Proc Natl Acad Sci U S A. 2011;108(18):7397-402. DOI:10.1073/pnas.1019266108 |
11. McCarthy PC, Saksena R, Peterson DC, Lee CH, An Y, Cipollo JF, and Vann WF. (2013). Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates. Glycoconj J. 2013;30(9):857-70. DOI:10.1007/s10719-013-9490-x |
12. Lizak C, Worrall LJ, Baumann L, Pfleiderer MM, Volkers G, Sun T, Sim L, Wakarchuk W, Withers SG, and Strynadka NCJ. (2017). X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid. Sci Rep. 2017;7(1):5842. DOI:10.1038/s41598-017-05627-z |

13. pmid= 8884739