New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities

Abstract

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Installing hydrolytic activity into a completely de novo protein framework

“…In our case, the reaction is turned over expelling acetate, and it is not driven by other nucleophilic side chains near the active site, which can become irreversibly acylated as the reaction proceeds contributing to observed kinetic profiles. 18,29 Indeed, even over a long time course and >6 turnovers, we observed only one small additional peak by Fig. 1.7-1), indicating limited secondary acylation.…”

“…For direct comparison to previous hydrolase designs, [17][18][19] we used a colorimetric p-nitrophenyl acetate (pNPA)-based assay to test for activity in the CC-Hept series. The single-mutant peptide assemblies, and hexameric CC-Hept-H-C-I showed no or little hydrolysis of pNPA above baseline at pH 7.0 (Fig.…”

“…Often only moderate efficiencies are achieved, although these can be further improved by directed evolution. [12][13][14][15][16] Computational approaches have also been used to design esterases, again with moderate activities being achieved through the placement of a histidine (His) residue into the active site of thioredoxin, 17 the C-terminal domain of calmodulin 18 and cysteine-His (Cys-His) dyads installed into existing / protein folds. 19 By contrast, much less has been demonstrated for the design of completely de novo catalysts; that is, where both the protein scaffold and the catalytic activity are built from scratch.…”

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

“…Indeed, a recent computational study pointed out how target reactivity can be one mutation away from a nonenzymatic protein (if well picked) [48]. Due to the scope of this book, we refer the reader interested in de novo design to recent studies on this topic [49].…”

“…Since the pioneering work [10,14,15] [46,47]. Despite the success of de novo design in providing novel structures and activity, its difficulties in achieving fast kinetics make it still preferable to modify templates available in Nature for the desired chemistry.Indeed, a recent computational study pointed out how target reactivity can be one mutation away from a nonenzymatic protein (if well picked) [48]. Due to the scope of this book, we refer the reader interested in de novo design to recent studies on this topic [49].…”