Restore the Activity and Stability of Nicotinamide Riboside Kinase by α-Helix Reconstruction and Semi-Rational Design

Area of Science:

• Biochemistry
• Enzyme Engineering
• Structural Biology

Background:

• Nicotinamide riboside kinase (NRK) is crucial for converting nicotinamide riboside (NR) to nicotinamide mononucleotide (NMN).
• Structural instability in NRKs limits their industrial utility.
• Developing methods to enhance NRK activity and stability is industrially important.

Purpose of the Study:

• To improve the activity and thermal stability of Kag-NRK from Kluyveromyces marxianus.
• To develop an alpha-helix reconstruction method for enzyme engineering.
• To identify specific mutations that enhance NRK function.

Main Methods:

• In silico screening of an alpha-helix library.
• Insertion of a 29-residue alpha-helix into Kag-NRK.
• Semi-rational design and molecular dynamics simulations.
• Characterization of enzyme activity and thermal stability.

Main Results:

• An engineered alpha-helix insertion between residues 55 and 56 significantly enhanced NRK activity and stability.
• The NRK variant W171V increased catalytic activity by 155% by enhancing flexibility.
• The NRK variant N214V increased thermal stability by 15.7°C and extended half-life.
• Molecular dynamics confirmed alpha-helix induced rigidity and substitution-mediated local stabilization.

Conclusions:

• Structural integrity is key for enzyme design and performance.
• Alpha-helix reconstruction and targeted mutations offer a viable strategy for enzyme improvement.
• This study provides a novel approach for enhancing NRK properties for industrial applications.