Circular permutation of Bacillus circulans xylanase: a kinetic and structural study

Area of Science:

• Enzymology
• Protein Engineering
• Structural Biology

Background:

• Bacillus circulans endoxylanase (Bcx) is a well-characterized enzyme with a beta-jellyroll fold.
• Circular permutation (CP) can alter protein termini and properties.
• CP of Bcx was explored to create novel enzyme variants.

Purpose of the Study:

• To generate and characterize circularly permuted variants of Bcx.
• To investigate the impact of CP on enzyme activity, structure, and stability.
• To establish a library of CP xylanases for further directed evolution.

Main Methods:

• Polymerase chain reaction (PCR) was used to create initial circular permutations.
• A library of CP Bcx variants was generated using random DNase cleavage.
• Screening for xylanase activity was performed on Congo Red-stained agar.
• Structural analysis included X-ray crystallography and NMR spectroscopy.

Main Results:

• 35 unique active circular permutants of Bcx were identified.
• New termini were found in both external loops and beta-strands.
• Some permutations placed catalytic residues near new termini, with one showing a 4-fold activity increase.
• CP caused minimal conformational changes but reduced thermal stability.
• Local mobility increased near altered termini.

Conclusions:

• Circular permutation is a viable strategy for creating active Bcx variants with altered properties.
• The generated library serves as a valuable resource for directed evolution and mechanistic studies.
• CP constructs facilitate intein-mediated replacement of catalytic residues with unnatural analogues.
• This work provides insights into enzyme engineering and glycoside hydrolysis mechanisms.