Increased Diels-Alderase activity through backbone remodeling guided by Foldit players

Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes(1-4). Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit(5,6) were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase(3) to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.