1. Molecular Modeling Of Endoglucanases August, 2007

2. INTRODUCTION

13. METHODS

16. Part 1 Homology Modeling

21. Structural-based Multiple Sequence Alignment

23. Alignment of the endoglucanases sequences from Acidothermus cellulolyticus (1ece) [358 residues] and Pyrococcus horikoshii (EGPh) [458 residues] with ClustalW. Extra loop?

25. EGPh Model loop loop

26. Alignment from MODELLER (Align2d method)

28. Secondary Structure Prediction by PSIDPRED

34. Z-score: -8.48 EGAc crystal structure (pdb code:1ece) EGPh model Z-score: -8.17

35. https://prosa.services.came.sbg.ac.at/prosa.php EGAc crystal structure (pdb code:1ece) EGPh model

36. Z-score (68.pdb) -8.17

37. Z-score (after 2ns MD) -9.1

38. Z-score(after 2ns&Bmin) -9.32

39. DOPE profile before and after MD

41. 100% 314 Total 37+23+2 Gly+Pro+end residues 85.4% 2 68 Most favored regions 12.1% 35 Additional allowed regions 1.6% 5 Generously allowed regions 1.0% 3 Disallowed regions % # Residues in

44. Y 264 W 233 W 337 F 29 EGPh Y 245 W 213 W 319 F 29 EGAc

45. -2 -1 +1 +2

46. Nucleophile -E 282 E 162 – proton donor

49. Part 2 MD simulations of EGPh & EGAc

51. Preparation Obtain pdb file Step 1 Check pKa & add H atoms accordingly Step 2 Solvate the protein in a water box Step 3 Add ions (Na + or Cl - )

54. EGPh EGAc

60. Comparison of Amino Acids Composition in EGAc and EGPh Charged residues: 55 vs. 80 3.99 15 5.87 21 Ala (A) 5.85 22 6.42 23 Val (V) 6.65 25 5.31 19 Ile (I) 7.18 27 8.38 30 Leu (L) 1.86 7 1.40 5 Met (M) 6.12 23 5.87 21 Pro (P) Hydrophobic 4.79 18 3.35 12 Phe (F) 9.84 37 9.22 33 Gly (G) Residues Mol% # Mol% # EGPh (total 376aa) EGAc (total 358aa)

61. # 80 7.18 27 7.26 26 Asp (D) Charged 5.05 19 1.96 7 Glu (E) 2.66 10 3.63 13 Arg (R) 6.38 24 2.51 9 Lys (K) 6.12 23 6.70 24 Asn (N) 1.86 7 5.59 20 Gln (Q) 1.06 4 1.12 4 Cys (C) 4.79 18 7.26 26 Ser (S) 5.32 20 5.31 19 Tyr (Y) Polar 5.05 19 5.03 18 Trp (W) 5.32 20 6.15 22 Thr (T) 2.93 11 1.68 6 His (H) Residues Mol% # Mol% # EGPh (total 376aa) EGAc (total 358aa) # 55

63. Salt Bridges

65. References

66. Extras

69. 12 Families with endoglucanse activity Asp (experimental) Not known Glu Asp (experimental) Not known Glu (experimental) Glu (experimental) Glu (experimental) Glu (experimental) Glu (experimental) Asp (experimental) Glu (experimental) Catalytic Proton Donor Fold 7-fold β -propeller --- Fold ( α / α ) 6 --- Not available Fold ( β / α ) 8 Fold -jelly roll Fold ( α / α ) 6 Fold ( α / α ) 6 Fold -jelly roll --- Fold ( β / α ) 8 Fold Asp (experimental) I endoglucanase; etc 74 Not known -- endoglucanase 61 Not known I endoglucanase; cellobiohydrolase. 48 Clan M Asp (experimental) I endoglucanase. 45 Not known I endoglucanase; xyloglucanase 44 Glu (experimental) R mannanase; -1,3-xylanase 26 Clan A Glu (experimental) R endoglucanase; xyloglucan hydrolase ; -1,3-1,4-glucanase 12 Clan C Asp (experimental) I endoglucanase; cellobiohydrolase; -glucosidase 9 Asp (inferred) I Chitosanase; Cellulase; Licheninase; Endo-1,4--xylanase 8 Clan M Glu (experimental) R endoglucanase; reducing end-acting cellobiohydrolase 7 Clan B Asp (experimental) I Endoglucanase;cellobiohydrolase 6 Glu (experimental) R Cellulase (EC 3.2.1.4); Glucan 1,3--glucosidase (EC 3.2.1.58); Endo-1,4--xylanase (EC 3.2.1.8); Cellulose 1,4--cellobiosidase (EC 3.2.1.91), etc 5 Clan A Catalytic Nucleophile/Base Mechanism Known Activity CAZY Family

70. Ligand: cellutetraose Catalytic Site