Design of a halogen bond catalyzed DNA endonuclease

Area of Science:

• Biochemistry
• Enzyme Engineering
• Bio-organic Chemistry

Background:

• Biological catalysts, or enzymes, typically rely on metal ions like magnesium (Mg²⁺) for catalytic activity.
• Mouse endonuclease G (mEndoG) is a magnesium-dependent enzyme involved in DNA processing.
• Understanding cofactor function is crucial for designing novel biocatalysts.

Purpose of the Study:

• To investigate the feasibility of replacing the Mg²⁺ cofactor in mEndoG with a halogen bond (X-bond).
• To engineer a novel catalytic X-bonding enzyme (cX-Zyme) with altered catalytic properties.
• To elucidate the mechanism of X-bond mediated catalysis in the engineered enzyme.

Main Methods:

• Site-directed mutagenesis of mEndoG to introduce meta-halotyrosine residues.
• Enzyme activity assays under varying pH conditions.
• Metal chelator (EDTA) inactivation studies.
• Mutation studies and electrostatic potential (ESP) calculations.

Main Results:

• Engineered meta-halotyrosine-mEndoG (^XY-mEndoG) constructs exhibited both acid and base catalysis.
• The ^XY-mEndoG was resistant to EDTA inactivation at low pH, indicating X-bond mediated catalysis.
• Iodinated constructs showed higher activity than chlorinated ones, suggesting enhanced X-bond strength.
• A model involving a hydrogen bond (H-bond) enhanced X-bond was proposed and supported by computational and mutational data.

Conclusions:

• A functional X-bond can replace the Mg²⁺ cofactor in mEndoG, creating a cX-Zyme.
• The engineered enzyme's catalytic mechanism involves an H-bond enhanced X-bond, modulated by pH.
• This study demonstrates the successful engineering of an enzyme with an unnatural catalytic mechanism, expanding the scope of biocatalysis.