Arif Sercan ŞAHUTOĞLU, Hatice DUMAN, Sercan KARAV, Steven Alex FRESE

Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods

EndoBI-1 and EndoBI-2 are two endo-β-N-acetylglucosaminidase isoenzymes that cleave N-N’-diacetylchitobiosyl moieties found in various types of native N-glycans. These N-glycans are indigestible by human infants and adults due to the lack of responsible glycosyl hydrolases and they act as selective prebiotics for a probiotic microorganism, Bifidobacterium longum subsp. infantis, in the large intestine. The selectivity and the thermostability of EndoBI-1 and EndoBI-2 suggest that these enzymes may be useful for many scientific and industrial applications. In this study, the growing numbers of homologous sequences in different databases were exploited in a comparative approach to investigate structural properties of EndoBI-1 and EndoBI-2 enzymes. Moreover, the complete and partial homology models of these two enzymes were generated and evaluated. Selected models were used for docking studies of the plus subsite ligand of these enzymes for further understanding on the substrate selectivity of EndoBI enzymes.

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1. Ling Z, Suits MDL, Bingham RJ, Bruce NC, Davies GJ et al. The X-ray crystal structure of an Arthrobacter protophormiae Endo-β-Nacetylglucosaminidase reveals a (β/α)8 catalytic domain, two ancillary domains and active site residues key for transglycosylation activity. Journal of Molecular Biology 2009; 389 (1): 1-9. doi: 10.1016/j.jmb.2009.03.050
2. Fairbanks AJ. The ENGases: Versatile biocatalysts for the production of homogeneous: N-linked glycopeptides and glycoproteins. Chemical Society Reviews 2017; 46 (16): 5128-5146. doi: 10.1039/C6CS00897F
3. Karav S, Parc LA, de Moura Bell JMLN, Liu Y, Mills DA et al. Characterizing the release of bioactive N-glycans from dairy products by a novel endo-β-N-acetylglucosaminidase. Biotechnology Progress 2015; 31 (5): 1331-1339. doi: 10.1002/btpr.2135
4. Garrido D, Nwosu C, Ruiz-Moyano S, Aldredge D, German JB et al. Endo-β-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins. Molecular & Cellular Proteomics 2012; 11 (9): 775-785. doi: 10.1074/mcp.M112.018119
5. Karav S, Parc LA, de Moura Bell JMLN, Rouquié C, Mills DA et al. Kinetic characterization of a novel endo-beta-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans. Enzyme and Microbial Technology 2015; 77: 46-53. doi: 10.1016/j. enzmictec.2015.05.007
6. Parc LA, Karav S, de Moura Bell JMLN, Frese SA, Liu Y et al. A novel endo‐β‐N‐acetylglucosaminidase releases specific N‐glycans depending on different reaction conditions. Biotechnology Progress 2015; 31 (5): 1323-1330. doi: 10.1002/btpr.2133
7. Sjögren J, Collin M. Bacterial glycosidases in pathogenesis and glycoengineering. Future Microbiology 2014; 9 (9): 1039-1051. doi: 10.2217/fmb.14.71
8. Seki H, Huang Y, Arakawa T, Yamada C, Kinoshita T et al. Structural basis for the specific cleavage of core-fucosylated N-glycans by endoN-acetylglucosaminidase from the fungus Cordyceps militaris. Journal of Biological Chemistry 2019; 294 (45): 17143-17154. doi: 10.1074/ jbc.RA119.010842
9. Karlsson M, Stenlid J. Evolution of family 18 glycoside hydrolases: Diversity, domain structures and phylogenetic relationships. Journal of Molecular Microbiology and Biotechnology 2009; 16 (3-4): 208-223. doi: 10.1159/000151220
10. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology 1990; 215 (3): 403- 410. doi: 10.1016/S0022-2836(05)80360-2
11. McWilliam H, Li W, Uludag M, Squizzato S, Park YM et al. Analysis tool web services from the EMBL-EBI. Nucleic Acids Research 2013; 41 (W1): 597-600. doi: 10.1093/nar/gkt376
12. Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research 2014; 42 (W1): 320-324. doi: 10.1093/nar/gku316
13. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: A sequence logo generator. Genome Research 2004; 14 (6): 1188-1190. doi: 10.1101/gr.849004
14. Kim DE, Chivian D, Baker D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Research 2004; 32: 526- 531. doi: 10.1093/nar/gkh468
15. Rohl CA, Strauss CEM, Misura KMS, Baker D. Protein structure prediction using Rosetta. Methods in Enzymology 2004; 383: 66-93. doi: 10.1016/S0076-6879(04)83004-0
16. Roy A, Kucukural A, Zhang Y. I-TASSER: A unified platform for automated protein structure and function prediction. Nature Protocols 2010; 5 (4): 725-738. doi: 10.1038/nprot.2010.5
17. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography 1993; 26 (2): 283-291. doi: 10.1107/S0021889892009944
18. Hooft RWW, Vriend G, Sander C, Abola EE. Errors in protein structures. Nature 1996; 381 (6580): 272. doi: 10.1038/381272a0
19. Colovos C, Yeates TO. Verification of protein structures: Patterns of nonbonded atomic interactions. Protein Science 1993; 2 (9): 1511- 1519. doi: 10.1002/pro.5560020916
20. Lüthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature 1992; 356 (6364): 83-85. doi: 10.1038/356083a0
21. Bowie JU, Lüthy R, Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional stucture. Science 1991; 253 (5016): 164-170. doi: 10.1126/science.1853201
22. Pontius J, Richelle J, Wodak SJ. Deviations from standard atomic volumes as a quality measure for protein crystal structures. Journal of Molecular Biology 1996; 264 (1): 121-126. doi: 10.1006/jmbi.1996.0628
23. Humphrey W, Dalke A, Schulten K. VMD: Visual molecular dynamics. Journal of Molecular Graphics 1996; 14 (1): 33-38. doi: 10.1016/0263-7855(96)00018-5
24. Laskowski RA. PDBsum: summaries and analyses of PDB structures. Nucleic Acids Research 2001; 29 (1): 221-222. doi: 10.1093/ nar/29.1.221
25. Grosdidier A, Zoete V, Michielin O. SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Research 2011; 39 (SUPPL. 2): 270-277. doi: 10.1093/nar/gkr366
26. Grosdidier A, Zoete V, Michielin O. Fast docking using the CHARMM force field with EADock DSS. Journal of Computational Chemistry 2011; 32 (10): 2149-2159. doi: 10.1002/jcc.21797
27. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM et al. UCSF Chimera - A visualization system for exploratory research and analysis. Journal of Computational Chemistry 2004; 25 (13): 1605-1612. doi: 10.1002/jcc.20084
28. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E et al. Scalable molecular dynamics with NAMD. Journal of Computational Chemistry 2005; 26 (16): 1781-1802. doi: 10.1002/jcc.20289
29. Brooks BR, Brooks CL, Mackerell AD, Nilsson L, Petrella RJ et al. CHARMM: The biomolecular simulation program. Journal of Computational Chemistry 2009; 30 (10): 1545-1614. doi: 10.1002/jcc.21287
30. MacKerell AD, Bashford D, Bellot M, Dunbrack RL, Evanseck JD et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. The Journal of Physical Chemistry B 1998; 102 (18): 3586-3616. doi: 10.1021/jp973084f
31. Mackerell AD, Feig M, Brooks CL. Improved treatment of the protein backbone in empirical force fields. Journal of the American Chemical Society 2004; 126 (3): 698-699. doi: 10.1021/ja036959e
32. Price DJ, Brooks CL. A modified TIP3P water potential for simulation with Ewald summation. The Journal of Chemical Physics 2004; 121 (20): 10096-10103. doi: 10.1063/1.1808117
33. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H et al. A smooth particle mesh Ewald method. The Journal of Chemical Physics 1995; 103 (19): 8577-8593. doi: 10.1063/1.470117
34. Trastoy B, Lomino JV, Pierce BG, Carter LG, Gunther S et al. Crystal structure of Streptococcus pyogenes EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies. Proceedings of the National Academy of Sciences 2014; 111 (18): 6714-6719. doi: 10.1073/pnas.1322908111
35. Klontz EH, Trastoy B, Deredge D, Fields JK, Li C et al. Molecular basis of Broad spectrum N-glycan specificity and processing of therapeutic IgG monoclonal antibodies by Endoglycosidase S2. American Chemical Society Central Science 2019; 5: 524-538. doi: 10.1021/acscentsci.8b00917
36. Trastoy B, Klontz E, Orwenyo J, Marina A, Wang LX et al. Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S. Nature Communications 2018; 9 (1): 1874. doi: 10.1038/s41467-018-04300-x
37. Ficko-Blean E, Gregg KJ, Adams JJ, Hehemann JH, Czjzek M et al. Portrait of an enzyme, a complete structural analysis of a multimodular β-N-acetylglucosaminidase from Clostridium perfringens. Journal of Biological Chemistry 2009; 284 (15): 9876-9884. doi: 10.1074/jbc. M808954200
38. Abbott DW, Boraston A. Structural analysis of a putative family 32 carbohydrate-binding module from the Streptococcus pneumoniae enzyme EndoD. Acta Crystallographica Section F: Structural Biology and Crystallization Communications 2011; 67 (4): 429-433. doi: 10.1107/S1744309111001874
39. Abbott DW, Eirín-López JM, Boraston AB. Insight into ligand diversity and novel biological roles for family 32 carbohydrate-binding modules. Molecular Biology and Evolution 2008; 25 (1): 155-167. doi: 10.1093/molbev/msm243
40. McIntosh LP, Hand G, Johnson PE, Joshi MD, Korner M et al. The pK(a) of the general acid/base carboxyl group of a glycosidase cycles during catalysis: A 13C-NMR study of Bacillus circulans xylanase. Biochemistry 1996; 35 (31): 9958-9966. doi: 10.1021/bi9613234
41. Synstad B, Gåseidnes S, van Aalten DMF, Vriend G, Nielsen JE et al. Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase. European Journal of Biochemistry 2004; 271 (2): 253-262. doi: 10.1046/j.1432-1033.2003.03923.x
42. He Y, Macauley MS, Stubbs KA, Vocadlo DJ, Davies GJ. Visualizing the reaction coordinate of an O-GlcNAc hydrolase. Journal of the American Chemical Society 2010; 132 (6): 1807-1809. doi: 10.1021/ja9086769
43. Hennig M, Jansonius JN, van Scheltinga ACT, Dijkstra BW, Schlesier B. Crystal structure of concanavalin B at 1.65 Å resolution. An ‘inactivated’ chitinase from seeds of Canavalia ensiformis. Journal of Molecular Biology 1995; 254 (2): 237-246. doi: 10.1006/jmbi.1995.0614