Merging Peptide and Enzyme Catalysis in a Single Protein Domain Bearing Two Separated Active Sites

Abstract A single protein that catalyzes two different reactions at two distinct active sites within one structural domain may be beneficial for cascade reactions. We explored this approach by merging a tripeptide catalyst with an alcohol dehydrogenase as a case study to bring together the strengths of enzymatic redox chemistry and peptide‐catalyzed carbon–carbon bond formation. A proline‐based peptide catalyst for C─C bond formation relying on an enamine intermediate was successfully merged to the N‐terminus of an alcohol dehydrogenase to catalyze a cascade involving alcohol oxidation followed by C─C bond formation. It turned out that the reaction speed of the two catalytic sites diverged, and the ε‐amino group of lysine residues present in the enzyme interfered with the organocatalytic proline activity. Removing/exchanging lysine residues within the enzyme reduced the background reaction but also the native redox activity. Interestingly, exchanging the N‐terminal proline with a histidine switched the stereopreference. The simultaneous cascade reaction of alcohol oxidation to the aldehyde and C─C bond formation with nitrostyrene presents a first proof‐of‐concept for bringing peptide catalysis together with enzyme catalysis and creating a bi‐active site catalyst within a single domain.