Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Biotechnology

Background:

• Cofactor utilization is crucial for oxidoreductase-catalyzed biotransformations.
• Improving cofactor recycling is a key strategy for enhancing biocatalytic efficiency.
• Existing methods for cofactor regeneration often face limitations in efficiency and stability.

Purpose of the Study:

• To enhance cofactor recycling in cofactor-dependent biocatalysis.
• To improve the efficiency of coupled oxidoreductive reactions.
• To demonstrate a novel strategy for spatially programmed enzyme assembly.

Main Methods:

• Independent expression of glycerol dehydrogenase (GlyDH) and glutamate dehydrogenase (GluDH) fused with phage protein A*.
• Enzymatic cross-linking to link ssDNA to the fused enzymes.
• Assembly of enzyme-ssDNA conjugates into desired structures via DNA base pairing.
• Analysis of reaction efficiency and cofactor utilization using HPLC.

Main Results:

• Fusion with protein A* slightly improved specific enzyme activity.
• Cofactor utilization efficiency was significantly enhanced (23.8- to 41.9-folds) in the assembled system.
• Evidence of a substrate-channeling mechanism due to enzyme proximity was observed.
• Cofactor recycling efficiency showed architecture-dependent behavior based on enzyme stoichiometry.

Conclusions:

• Spatially programmed assembly of enzymes using ssDNA is a novel strategy for cofactor recycling.
• This approach significantly enhances the efficiency of coupled oxidoreductive biotransformations.
• The proximity effect and substrate channeling contribute to improved cofactor utilization.