A team of researchers designed retro-aldol enzymes using computational methods to catalyze the breaking of carbon-carbon bonds in nonnatural substrates. The study focused on creating enzymes that can perform a retro-aldol reaction, which involves breaking a carbon-carbon bond in a substrate not found in biological systems. The researchers used new algorithms that rely on hashing techniques to construct active sites for multistep reactions, and designed enzymes using four different catalytic motifs. Of the 72 experimentally characterized designs, 32 showed detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, compared to those involving charged side-chain networks. The study highlights the importance of accurate design in enzyme creation, as confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, which were nearly superimposable on the design model. The researchers also tested the accuracy of their design models by solving the structures of two of the designs by x-ray crystallography, which showed that the designed catalytic residues superimposed well on the original design model. The study demonstrates that computational design of enzymes can be achieved, and that the catalytic strategies most accessible to nascent enzymes are those that involve explicit water molecules for proton shuffling. However, the catalytic proficiency of the designed enzymes is still far from that of naturally occurring enzymes, which have a k_cat/K_M of about 1 M⁻¹ s⁻¹. The researchers also found that the very low k_cat value is probably associated with low reactivity of the imine-forming lysine. The study also highlights the challenges of computational design of extended polar networks, as well as the versatility of bound water molecules, which can readily reorient to switch between acting as hydrogen-bond acceptors and donors. The researchers suggest that computationally designed enzymes may resemble primordial enzymes more than they resemble highly refined modern-day enzymes, as the ability to design only three to four catalytic residues parallels the infinitesimal probability that, early in evolution, more than three to four residues would have happened to be positioned appropriately for catalysis. The study concludes that while the results demonstrate that novel enzyme activities can be designed from scratch, there is still a significant gap between the activities of the designed catalysts and those of naturally occurring enzymes. The researchers suggest that future work should focus on incorporating additional features into the design process to achieve catalytic activities approaching those of naturally occurring enzymes. The close agreement between the two crystal structures and the design models gives credence to the strategy of testing hypotheses about catalytic mechanisms by generating and testing the corresponding designs.A team of researchers designed retro-aldol enzymes using computational methods to catalyze the breaking of carbon-carbon bonds in nonnatural substrates. The study focused on creating enzymes that can perform a retro-aldol reaction, which involves breaking a carbon-carbon bond in a substrate not found in biological systems. The researchers used new algorithms that rely on hashing techniques to construct active sites for multistep reactions, and designed enzymes using four different catalytic motifs. Of the 72 experimentally characterized designs, 32 showed detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, compared to those involving charged side-chain networks. The study highlights the importance of accurate design in enzyme creation, as confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, which were nearly superimposable on the design model. The researchers also tested the accuracy of their design models by solving the structures of two of the designs by x-ray crystallography, which showed that the designed catalytic residues superimposed well on the original design model. The study demonstrates that computational design of enzymes can be achieved, and that the catalytic strategies most accessible to nascent enzymes are those that involve explicit water molecules for proton shuffling. However, the catalytic proficiency of the designed enzymes is still far from that of naturally occurring enzymes, which have a k_cat/K_M of about 1 M⁻¹ s⁻¹. The researchers also found that the very low k_cat value is probably associated with low reactivity of the imine-forming lysine. The study also highlights the challenges of computational design of extended polar networks, as well as the versatility of bound water molecules, which can readily reorient to switch between acting as hydrogen-bond acceptors and donors. The researchers suggest that computationally designed enzymes may resemble primordial enzymes more than they resemble highly refined modern-day enzymes, as the ability to design only three to four catalytic residues parallels the infinitesimal probability that, early in evolution, more than three to four residues would have happened to be positioned appropriately for catalysis. The study concludes that while the results demonstrate that novel enzyme activities can be designed from scratch, there is still a significant gap between the activities of the designed catalysts and those of naturally occurring enzymes. The researchers suggest that future work should focus on incorporating additional features into the design process to achieve catalytic activities approaching those of naturally occurring enzymes. The close agreement between the two crystal structures and the design models gives credence to the strategy of testing hypotheses about catalytic mechanisms by generating and testing the corresponding designs.