3WP5
The crystal structure of mutant CDBFV E109A from Neocallimastix patriciarum
Summary for 3WP5
| Entry DOI | 10.2210/pdb3wp5/pdb |
| Related | 3WP4 3WP6 |
| Descriptor | CDBFV (2 entities in total) |
| Functional Keywords | xylanase, regulatory n-terminal region, disulfide bond, industrial enzyme, beta-jellyroll fold, hydrolase |
| Biological source | Neocallimastix patriciarum |
| Total number of polymer chains | 1 |
| Total formula weight | 24635.00 |
| Authors | Cheng, Y.S.,Chen, C.C.,Huang, C.H.,Huang, T.Y.,Ko, T.P.,Huang, J.W.,Wu, T.H.,Liu, J.R.,Guo, R.T. (deposition date: 2014-01-09, release date: 2014-03-19, Last modification date: 2024-11-13) |
| Primary citation | Cheng, Y.S.,Chen, C.C.,Huang, C.H.,Ko, T.P.,Luo, W.,Huang, J.W.,Liu, J.R.,Guo, R.T. Structural analysis of a glycoside hydrolase family 11 xylanase from Neocallimastix patriciarum: insights into the molecular basis of a thermophilic enzyme. J.Biol.Chem., 289:11020-11028, 2014 Cited by PubMed Abstract: The catalytic domain of XynCDBFV, a glycoside hydrolase family 11 (GH11) xylanase from ruminal fungus Neocallimastix patriciarum previously engineered to exhibit higher specific activity and broader pH adaptability, holds great potential in commercial applications. Here, the crystal structures of XynCDBFV and its complex with substrate were determined to 1.27-1.43 Å resolution. These structures revealed a typical GH11 β-jelly-roll fold and detailed interaction networks between the enzyme and ligands. Notably, an extended N-terminal region (NTR) consisting of 11 amino acids was identified in the XynCDBFV structure, which is found unique among GH11 xylanases. The NTR is attached to the catalytic core by hydrogen bonds and stacking forces along with a disulfide bond between Cys-4 and Cys-172. Interestingly, the NTR deletion mutant retained 61.5% and 19.5% enzymatic activity at 55 °C and 75 °C, respectively, compared with the wild-type enzyme, whereas the C4A/C172A mutant showed 86.8% and 23.3% activity. These results suggest that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-172 disulfide bond is critical to the NTR-mediated interactions. Furthermore, we also demonstrated that Pichia pastoris produces XynCDBFV with higher catalytic activity at higher temperature than Escherichia coli, in which incorrect NTR folding and inefficient disulfide bond formation might have occurred. In conclusion, these structural and functional analyses of the industrially favored XynCDBFV provide a molecular basis of NTR contribution to its thermostability. PubMed: 24619408DOI: 10.1074/jbc.M114.550905 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.32 Å) |
Structure validation
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