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Expression, purification, and characterization of a new Glucosyltransferase involved in the third step of O-antigen repeating-unit biosynthesis of Escherichia coli O152

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Abstract

The O antigen is indispensable for the full function and virulence of pathogenic bacteria. During O-repeating unit (RU) biosynthesis, committed glycosyltransferases (GTs) transfer various sugars from an activated sugar donor to the appropriate lipid carrier sequentially. While the nucleotide sequence specific for O antigen of pathogenic bacteria is already known, the exact substrate specificity of most hypothetical GTs have yet be characterized. In the present paper, we report the biochemical characterization of one alpha-glucosyltransferase, WfgE, a member of GT family 4. This enzyme is implicated in the pentasaccharide RU biosynthetic pathway of E. coli O152 O antigen. A chemoenzymatically synthesized acceptor (GlcGlcNAc α-PP-O(CH2)10CH3) was used to characterize the WfgE activity. The enzyme product was determined to have a 1,2-linkage using strategy based on collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). The lack of a DxD motif and its high activity without divalent metal ions suggests that WfgE belongs to the GT-B fold superfamily. The enzyme is specific for beta-glucose or galactose-terminating acceptor substrates, and in particular UDP-glucose but also UDP-galactose as donor substrates. Our results suggest that WfgE catalyses the addition of the third sugar residue of the E. coli O152 O-RU. The recombinant GST-WfgE was solubilized and further purified to homogeneity via GST affinity chromatography, paving the way for structure-function relationship studies.

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References

  1. Hug, I., Feldman, M.F.: Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria. Glycobiology. 21, 138–151 (2011). https://doi.org/10.1093/glycob/cwq148

    Article  CAS  PubMed  Google Scholar 

  2. Cheasty, T., Rowe, B.: Antigenic relationships between the enteroinvasive Escherichia coli O antigens O28ac, O112ac, O124, O136, O143, O144, O152, and O164 and Shigella O antigens. J. Clin. Microbiol. 17, 681–684 (1983)

    Article  CAS  Google Scholar 

  3. Olsson, U., Lycknert, K., Stenutz, R., Weintraub, A., Widmalm, G.: Structural analysis of the O-antigen polysaccharide from Escherichia coli O152. Carbohydr. Res. 340, 167–171 (2005). https://doi.org/10.1016/j.carres.2004.11.008

    Article  CAS  PubMed  Google Scholar 

  4. Brockhausen, I., Hu, B., Liu, B., Lau, K., Szarek, W.A., Wang, L., Feng, L.: Characterization of two beta-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152. J. Bacteriol. 190, 4922–4932 (2008). https://doi.org/10.1128/JB.00160-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Xu, C., Liu, B., Hu, B., Han, Y., Feng, L., Allingham, J.S., Szarek, W.A., Wang, L., Brockhausen, I.: Biochemical characterization of UDP-gal:GlcNAc-pyrophosphate-lipid beta-1,4-Galactosyltransferase WfeD, a new enzyme from Shigella boydii type 14 that catalyzes the second step in O-antigen repeating-unit synthesis. J. Bacteriol. 193, 449–459 (2011). https://doi.org/10.1128/JB.00737-10

    Article  CAS  PubMed  Google Scholar 

  6. Wang, S., Czuchry, D., Liu, B., Vinnikova, A., Gao, Y., Vlahakis, J.Z., Szarek, W.A., Wang, L., Feng, L., Brockhausen, I.: Characterization of UDP-gal: GalNAc alpha-PP-R beta 1,3-gal-transferase WbwC, a T-synthase from STEC O104. Glycobiology. 23, 1371–1371 (2013)

    Article  Google Scholar 

  7. Riley, J.G., Menggad, M., Montoya-Peleaz, P.J., Szarek, W.A., Marolda, C.L., Valvano, M.A., Schutzbach, J.S., Brockhausen, I.: The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide. Glycobiology. 15, 605–613 (2005). https://doi.org/10.1093/glycob/cwi038

    Article  CAS  PubMed  Google Scholar 

  8. Gao, Y., Liu, B., Strum, S., Schutzbach, J.S., Druzhinina, T.N., Utkina, N.S., Torgov, V.I., Danilov, L.L., Veselovsky, V.V., Vlahakis, J.Z., Szarek, W.A., Wang, L., Brockhausen, I.: Biochemical characterization of WbdN, a beta1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157. Glycobiology. 22, 1092–1102 (2012). https://doi.org/10.1093/glycob/cws081

    Article  CAS  PubMed  Google Scholar 

  9. Han, W.Q., Cai, L., Wu, B.L., Li, L., Xiao, Z.Y., Cheng, J.S., Wang, P.G.: The wciN gene encodes an alpha-1,3-Galactosyltransferase involved in the biosynthesis of the capsule repeating unit of Streptococcus pneumoniae serotype 6B. Biochemistry-Us. 51, 5804–5810 (2012). https://doi.org/10.1021/bi300640b

    Article  CAS  Google Scholar 

  10. Sambrook, J., Fritsch, E. F., Maniatis, T.: Molecular Cloning: A Laboratory Manual, 2nd edn, pp. 201–203. Cold Spring Harbor Laboratory Press, New York, (1989)

  11. Kaniuk, N.A., Vinogradov, E., Li, J.J., Monteiro, M.A., Whitfield, C.: Chromosomal and plasmid-encoded enzymes are required for assembly of the R3-type core oligosaccharide in the lipopolysaccharide of Escherichia coli O157 : H7. J. Biol. Chem. 279, 31237–31250 (2004). https://doi.org/10.1074/jbc.M401879200

    Article  CAS  PubMed  Google Scholar 

  12. Shibayama, K., Ohsuka, S., Sato, K., Yokoyama, K., Horii, T., Ohta, M.: Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR. FEMS Microbiol. Lett. 174, 105–109 (1999). https://doi.org/10.1111/j.1574-6968.1999.tb13555.x

    Article  CAS  PubMed  Google Scholar 

  13. Burda, P., Aebi, M.: The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation. Glycobiology. 8, 455–462 (1998). https://doi.org/10.1093/glycob/8.5.455

    Article  CAS  PubMed  Google Scholar 

  14. Shao, J., Li, M., Jia, Q., Lu, Y.Q., Wang, P.G.: Sequence of Escherichia coli O128 antigen biosynthesis cluster and functional identification of an alpha-1,2-fucosyltransferase. FEBS Lett. 553, 99–103 (2003). https://doi.org/10.1016/S0014-5793(03)00980-3

    Article  CAS  PubMed  Google Scholar 

  15. Salinas, S.R., Bianco, M.I., Barreras, M., Ielpi, L.: Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose: glycolipid 4-beta-D-mannosyltransferase from Xanthomonas campestris pv. campestris. Glycobiology. 21, 903–913 (2011). https://doi.org/10.1093/glycob/cwr022

    Article  CAS  PubMed  Google Scholar 

  16. Schmidt, H., Hansen, G., Singh, S., Hanuszkiewicz, A., Lindner, B., Fukase, K., Woodard, R.W., Holst, O., Hilgenfeld, R., Mamat, U., Mesters, J.R.: Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis. P. Natl. Acad. Sci. USA. 109, 6253–6258 (2012). https://doi.org/10.1073/pnas.1119894109

    Article  Google Scholar 

  17. Mengin-Lecreulx, D., Texier, L., Rousseau, M., van Heijenoort, J.: The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis. J. Bacteriol. 173, 4625–4636 (1991). https://doi.org/10.1128/jb.173.15.4625-4636.1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Geremia, R.A., Roux, M., Ferreiro, D.U., Dauphin-Dubois, R., Lellouch, A.C., Ielpi, L.: Expression and biochemical characterisation of recombinant AceA, a bacterial alpha-mannosyltransferase. Mol. Gen. Genet. 261, 933–940 (1999). https://doi.org/10.1007/s004380051040

    Article  CAS  PubMed  Google Scholar 

  19. Kanipes, M.I., Kalb, S.R., Cotter, R.J., Hozbor, D.F., Lagares, A., Raetz, C.R.H.: Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the rhizobium leguminosarum mannosyl transferase LpcC - role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants. J. Biol. Chem. 278, 16365–16371 (2003). https://doi.org/10.1074/jbc.M301256200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Losey, H.C., Peczuh, M.W., Chen, Z., Eggert, U.S., Dong, S.D., Pelczer, I., Kahne, D., Walsh, C.T.: Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides. Biochemistry. 40, 4745–4755 (2001). https://doi.org/10.1021/bi010050w

    Article  CAS  PubMed  Google Scholar 

  21. Takeuchi, H., Fernandez-Valdivia, R.C., Caswell, D.S., Nita-Lazar, A., Rana, N.A., Garner, T.P., Weldeghiorghis, T.K., Macnaughtan, M.A., Jafar-Nejad, H., Haltiwanger, R.S.: Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase. P. Natl. Acad. Sci. USA. 108, 16600–16605 (2011). https://doi.org/10.1073/pnas.1109696108

    Article  Google Scholar 

  22. Ono, E., Homma, Y., Horikawa, M., Kunikane-Doi, S., Imai, H., Takahashi, S., Kawai, Y., Ishiguro, M., Fukui, Y., Nakayama, T.: Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera). Plant Cell. 22, 2856–2871 (2010). https://doi.org/10.1105/tpc.110.074625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Aharoni, A., Gaidukov, L., Khersonsky, O., Gould, S.M., Roodveldt, C., Tawfik, D.S.: The 'evolvability' of promiscuous protein functions. Nat. Genet. 37, 73–76 (2005). https://doi.org/10.1038/Ng1482

    Article  CAS  PubMed  Google Scholar 

  24. Bloom, J.D., Arnold, F.H.: In the light of directed evolution: pathways of adaptive protein evolution. P. Natl. Acad. Sci. USA. 106, 9995–10000 (2009). https://doi.org/10.1073/pnas.0901522106

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Fundamental Research Funds for the Central Universities, Nankai University (63191133) and Tianjin enterprise science and technology commissioner project (18JCTPJC65600).

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Authors and Affiliations

  1. Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, TEDA College, Nankai University, Tianjin, People’s Republic of China

    Chenying Dong, Diange Li, Ru Wang & Dawei Zhou

  2. TEDA Institue of Biological Sciences and Biotechnology, Nankai University, #23 HongDa Street, TEDA, Tianjin, 300457, China

    Chenying Dong, Diange Li, Ru Wang & Dawei Zhou

  3. Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin, People’s Republic of China

    Chenying Dong, Diange Li, Ru Wang & Dawei Zhou

  4. Tianjin Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, Tianjin, People’s Republic of China

    Chenying Dong, Diange Li, Ru Wang & Dawei Zhou

  5. School of Automation and Electrical Engineering, Tianjin University of Technology and Education, Tianjin, People’s Republic of China

    Jian Chu

  6. Department of Neurology, Tianjin First Center Hospital, Tianjin, People’s Republic of China

    Zhongying Gong

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  1. Chenying Dong
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Dong, C., Li, D., Wang, R. et al. Expression, purification, and characterization of a new Glucosyltransferase involved in the third step of O-antigen repeating-unit biosynthesis of Escherichia coli O152. Glycoconj J 37, 139–149 (2020). https://doi.org/10.1007/s10719-020-09907-1

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