A Genetically Encoded Redox-Active Nicotinamide Amino Acid

Abstract

Nicotinamide-containing cofactors play an essential role in many enzymes that catalyze two-electron redox reactions. However, it is difficult to engineer nicotinamide binding sites into proteins due to the extended nature of the cofactor-protein interface and the precise orientation of the nicotinamide moiety required for efficient electron transfer to or from the substrate. To address these challenges, we genetically encoded a noncanonical amino acid (ncAA) bearing a nicotinamide side chain in bacteria. This redox-active amino acid, termed Nic1, exhibits similar electrochemical properties to the natural cofactor nicotinamide adenine dinucleotide (NAD⁺). Nic1 can be reversibly reduced and oxidized using chemical reagents both free in solution and when incorporated into a model protein. This genetically encodable cofactor can be introduced into proteins in a site-specific fashion and may serve as a tool to study electron-transfer mechanisms in enzymes and to engineer redox-active proteins.

Figure 1.

Redox properties of Nic1. A) Comparison of the chemical structure of Nic1 and the natural cofactor Nicotinamide Adenine Dinucleotide (NAD⁺) with the nicotinamide ring highlighted in blue. B) Cyclic voltammogram of NAD⁺ (dashed) and K-Nic1 (solid). C) Absorbance spectra of K-Nic1 (dashed), after its reduction with NaBH₄ (solid), and followed by oxidation with Cu(II)/H₂O₂ (dotted).

Figure 2.

Characterization of Nic1-containing proteins. A) sfGFP expression in the presence of 2.5 mM lysine or 2.5 mM K-Nic1, with and without 0.5% casamino acids (CA) added to M9 expression media. B) LC-MS analysis of sfGFP Y151Nic1. C) Absorbance spectra of MBP variants following purification. D) NaBH₄ titration of MBP T81Nic1 in 10 increments, shown as thin lines. E) Absorbance spectra of two redox cycles of MBP T81Nic1.

Scheme 1.

Synthesis of K-Nic1.