Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease

Area of Science:

• Molecular Biology
• Enzymology
• Protein Science

Background:

• Homing endonucleases are mobile genetic elements crucial for gene transfer.
• Understanding I-CeuI function is vital for its application in genetic engineering and gene therapy.
• Identifying critical residues informs protein design and functional studies.

Purpose of the Study:

• To identify critical amino acid residues for the catalytic activity and DNA recognition of the I-CeuI homing endonuclease.
• To investigate the roles of conserved motifs, specifically the LAGL I-DADG dodecapeptide and TQH sequence, in enzyme function.

Main Methods:

• Comparative sequence analysis of homologous proteins to predict functional residues.
• Genetic selection in Escherichia coli to generate enzyme variants with altered function.
• In vitro kinetic analysis (Km and kcat) of wild-type and variant I-CeuI endonucleases.

Main Results:

• Identified 37 amino acid positions, many clustered in conserved motifs, critical for I-CeuI function.
• Generated 49 variants, with several showing complete loss or significant reduction of endonuclease activity.
• One variant exhibited a threefold increase in top-strand cleavage efficiency compared to the wild-type enzyme.
• Substitutions in the TQH motif and residues outside the LAGL I-DADG motif impacted catalytic activity.

Conclusions:

• Amino acids within the LAGL I-DADG dodecapeptide are confirmed as functionally critical for I-CeuI activity.
• Residues outside the canonical LAGL I-DADG motif, including those in the TQH sequence, directly participate in catalysis.
• This study provides insights into the structure-function relationship of homing endonucleases, aiding future protein engineering efforts.