Format

Send to

Choose Destination
See comment in PubMed Commons below
J Biol Chem. 2014 Apr 18;289(16):11020-8. doi: 10.1074/jbc.M114.550905. Epub 2014 Mar 11.

Structural analysis of a glycoside hydrolase family 11 xylanase from Neocallimastix patriciarum: insights into the molecular basis of a thermophilic enzyme.

Author information

1
From the Genozyme biotechnology Inc., Taipei 106, Taiwan.

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.

KEYWORDS:

Crystal Structure; Disulfide Bond; Enzyme Mutation; Enzyme Structure; Glycoside Hydrolases; Industrial Enzyme; N-terminal Region; X-ray Crystallography; Xylanase

PMID:
24619408
PMCID:
PMC4036243
DOI:
10.1074/jbc.M114.550905
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Support Center