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.