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Crystal Structure of LysB4, an Endolysin from Bacillus cereus-Targeting Bacteriophage B4

  • Hong, Seokho (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University) ;
  • Son, Bokyung (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University) ;
  • Ryu, Sangryeol (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University) ;
  • Ha, Nam-Chul (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University)
  • Received : 2018.09.20
  • Accepted : 2018.11.11
  • Published : 2019.01.31

Abstract

Endolysins are bacteriophage-derived enzymes that hydrolyze the peptidoglycan of host bacteria. Endolysins are considered to be promising tools for the control of pathogenic bacteria. LysB4 is an endolysin produced by Bacillus cereus-infecting bacteriophage B4, and consists of an N-terminal enzymatic active domain (EAD) and a C-terminal cell wall binding domain (CBD). LysB4 was discovered for the first time as an L-alanoyl-D-glutamate endopeptidase with the ability to breakdown the peptidoglycan among B. cereus-infecting phages. To understand the activity of LysB4 at the molecular level, this study determined the X-ray crystal structure of the LysB4 EAD, using the full-length LysB4 endolysin. The LysB4 EAD has an active site that is typical of LAS-type enzymes, where $Zn^{2+}$ is tetrahedrally coordinated by three amino acid residues and one water molecule. Mutational studies identified essential residues that are involved in lytic activity. Based on the structural and biochemical information about LysB4, we suggest a ligand-docking model and a putative endopeptidase mechanism for the LysB4 EAD. These suggestions add insight into the molecular mechanism of the endolysin LysB4 in B. cereus-infecting phages.

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Fig. 1. The Structure of the LysB4 EAD.

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Fig. 2. Structural comparison to Ply500 EAD.

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Fig. 3. Ligand docking model with GlcNAc-MurNAc-LAla-D-Glu-meso-DAP-D-Ala.

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Fig. 4. The CD profiles and a putative endopeptidase mechanism of LysB4.

Table 1. X-ray diffraction and refinement statistics

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Table 2. Endopeptidase activity assay against several bacteria

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Acknowledgement

Supported by : National Research Foundation of Korea, Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET)

References

  1. Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213-221. https://doi.org/10.1107/S0907444909052925
  2. Asensio, J.L., Arda, A., Canada, F.J., and Jimenez-Barbero, J. (2013). Carbohydrate-aromatic interactions. Acc. Chem. Res. 46, 946-954. https://doi.org/10.1021/ar300024d
  3. Bochtler, M., Odintsov, S.G., Marcyjaniak, M., and Sabala, I. (2004). Similar active sites in lysostaphins and D-Ala-D-Ala metallopeptidases. Protein Sci. 13, 854-861. https://doi.org/10.1110/ps.03515704
  4. Borysowski, J., Weber-Dabrowska, B., and Gorski, A. (2006). Bacteriophage endolysins as a novel class of antibacterial agents. Exp. Biol. Med. 231, 366-377. https://doi.org/10.1177/153537020623100402
  5. Bussiere, D.E., Pratt, S.D., Katz, L., Severin, J.M., Holzman, T., and Park, C.H. (1998). The structure of VanX reveals a novel aminodipeptidase involved in mediating transposon-based vancomycin resistance. Mol. Cell 2, 75-84. https://doi.org/10.1016/S1097-2765(00)80115-X
  6. Dziarski, R., and Gupta, D. (2006). The peptidoglycan recognition proteins (PGRPs). Genome Biol. 7, 232. https://doi.org/10.1186/gb-2006-7-8-232
  7. Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486-501. https://doi.org/10.1107/S0907444910007493
  8. Etobayeva, I., Linden, S.B., Alem, F., Harb, L., Rizkalla, L., Mosier, P.D., Johnson, A.A., Temple, L., Hakami, R.M., and Nelson, D.C. (2018). Discovery and biochemical characterization of PlyP56, PlyN74, and PlyTB40-Bacillus specific endolysins. Viruses 10, 276. https://doi.org/10.3390/v10050276
  9. Fukushima, T., Yao, Y., Kitajima, T., Yamamoto, H., and Sekiguchi, J. (2007). Characterization of new L, D-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis. Mol. Genet. Genomics 278, 371-383. https://doi.org/10.1007/s00438-007-0255-8
  10. Ha, N.C., Oh, B.C., Shin, S., Kim, H.J., Oh, T.K., Kim, Y.O., Choi, K.Y., and Oh, B.H. (2000). Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states. Nat. Struct. Biol. 7, 147-153. https://doi.org/10.1038/72421
  11. Jang, Y., Choi, G., Hong, S., Jo, I., Ahn, J., Choi, S.H., and Ha, N.C. (2018). A novel tetrameric assembly configuration in VV2_1132, a LysR-type transcriptional regulator in Vibrio vulnificus. Mol. Cells 41, 301-310.
  12. Jones, P., Binns, D., Chang, H.Y., Fraser, M., Li, W., McAnulla, C., McWilliam, H., Maslen, J., Mitchell, A., Nuka, G., et al. (2014). InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236-1240. https://doi.org/10.1093/bioinformatics/btu031
  13. Kamisango, K., Saiki, I., Tanio, Y., Okumura, H., Araki, Y., Sekikawa, I., Azuma, I., and Yamamura, Y. (1982). Structures and biological activities of peptidoglycans of Listeria monocytogenes and Propionibacterium acnes. J. Biochem. 92, 23-33. https://doi.org/10.1093/oxfordjournals.jbchem.a133918
  14. Korndorfer, I.P., Kanitz, A., Danzer, J., Zimmer, M., Loessner, M.J., and Skerra, A. (2008). Structural analysis of the L-alanoyl-Dglutamate endopeptidase domain of Listeria bacteriophage endolysin Ply500 reveals a new member of the LAS peptidase family. Acta Crystallogr. D. Biol. Crystallogr. 64, 644-650. https://doi.org/10.1107/S0907444908007890
  15. Lee, J.H., Shin, H., Son, B., Heu, S., and Ryu, S. (2013). Characterization and complete genome sequence of a virulent bacteriophage B4 infecting food-borne pathogenic Bacillus cereus. Arch. Virol. 158, 2101-2108. https://doi.org/10.1007/s00705-013-1719-2
  16. Leive, L. (1968). Studies on the permeability change produced in coliform bacteria by ethylenediaminetetraacetate. J. Biol. Chem. 243, 2373-2380.
  17. Lim, J.H., Kim, M.S., Kim, H.E., Yano, T., Oshima, Y., Aggarwal, K., Goldman, W.E., Silverman, N., Kurata, S., and Oh, B.H. (2006). Structural basis for preferential recognition of diaminopimelic acidtype peptidoglycan by a subset of peptidoglycan recognition proteins. J. Biol. Chem. 281, 8286-8295. https://doi.org/10.1074/jbc.M513030200
  18. Loessner, M.J. (2005). Bacteriophage endolysins-current state of research and applications. Curr. Opin. Microbiol. 8, 480-487. https://doi.org/10.1016/j.mib.2005.06.002
  19. Loessner, M.J., Wendlinger, G., and Scherer, S. (1995). Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. Mol. Microbiol. 16, 1231-1241. https://doi.org/10.1111/j.1365-2958.1995.tb02345.x
  20. McCafferty, D.G., Lessard, I.A., and Walsh, C.T. (1997). Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX. Biochem. 36, 10498-10505. https://doi.org/10.1021/bi970543u
  21. McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Crystallogr. 40, 658-674. https://doi.org/10.1107/S0021889807021206
  22. Nelson, D.C., Schmelcher, M., Rodriguez-Rubio, L., Klumpp, J., Pritchard, D.G., Dong, S., and Donovan, D.M. (2012). Endolysins as antimicrobials. In Adv. Virus Res. (Elsevier), pp. 299-365.
  23. Nelson, M.D., and Fitch, D.H. (2012). Overlap extension PCR: an efficient method for transgene construction. In Molecular Methods for Evolutionary Genetics (Springer), pp. 459-470.
  24. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326.
  25. Owen, R.A., Fyfe, P.K., Lodge, A., Biboy, J., Vollmer, W., Hunter, W.N., and Sargent, F. (2018). Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens. Biochem. J. 475, 415-428. https://doi.org/10.1042/BCJ20170633
  26. Schleifer, K.H., and Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36, 407-477.
  27. Schmelcher, M., Donovan, D.M., and Loessner, M.J. (2012). Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 7, 1147-1171. https://doi.org/10.2217/fmb.12.97
  28. Son, B., Yun, J., Lim, J.A., Shin, H., Heu, S., and Ryu, S. (2012). Characterization of LysB4, an endolysin from the Bacillus cereusinfecting bacteriophage B4. BMC Microbiol. 12, 33. https://doi.org/10.1186/1471-2180-12-33
  29. Vagin, A., and Teplyakov, A. (2010). Molecular replacement with MOLREP. Acta Crystallogr. D. Biol. Crystallogr. 66, 22-25.
  30. Vollmer, W., Blanot, D., and de Pedro, M.A. (2008). Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149-167. https://doi.org/10.1111/j.1574-6976.2007.00094.x
  31. Winn, M.D., Ballard, C.C., Cowtan, K.D., Dodson, E.J., Emsley, P., Evans, P.R., Keegan, R.M., Krissinel, E.B., Leslie, A.G., McCoy, A., et al. (2011). Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr. 67, 235-242. https://doi.org/10.1107/S0907444910045749