A Genetically Encoded Redox-Active Nicotinamide Amino Acid.

Area of Science:

• Biochemistry
• Synthetic Biology
• Protein Engineering

Background:

• Nicotinamide cofactors are crucial for enzymatic two-electron redox reactions.
• Engineering proteins to bind nicotinamide cofactors is challenging due to complex interfaces and precise orientation requirements.

Purpose of the Study:

• To genetically encode a novel nicotinamide-containing amino acid (Nic1) for protein incorporation.
• To develop a tool for studying enzyme electron-transfer mechanisms and engineering redox-active proteins.

Main Methods:

• Genetic encoding of a noncanonical amino acid (ncAA) with a nicotinamide side chain in bacteria.
• Characterization of the electrochemical properties of the incorporated Nic1.
• Demonstration of reversible redox activity of Nic1 in solution and within a model protein.

Main Results:

• Successfully engineered and expressed a redox-active amino acid, Nic1, in bacteria.
• Nic1 demonstrated electrochemical properties similar to nicotinamide adenine dinucleotide (NAD+).
• Site-specific incorporation of Nic1 into a model protein enabled reversible redox reactions.

Conclusions:

• Genetically encoded Nic1 serves as a functional, site-specific cofactor mimic.
• Nic1 provides a novel tool for investigating enzyme mechanisms and engineering redox-active proteins.