research advances
September 2008 technical highlight
Designing activity
PSI-SGKB [doi:10.1038/th_psisgkb.2008.6]
The crystal structure of the most active of the modeled enzymes is shown in ribbon representation
The increasing ease in use of molecular biology techniques in recent years has contributed to the growth of the enzyme design field, where naturally occurring protein catalysts have been optimized to have improved stability and catalytic efficiency, and, for certain industrial processes, altered substrate selectivity. For the most part, enzyme design has taken the shape of directed evolution, in which large-scale random mutagenesis is coupled to selective screening for optimized catalytic activity of a given chemical reaction. While directed evolution requires no knowledge of active site structure and mechanism to be effective, the much-less successful rational design approach makes use of known structural organization of a catalytic site to purposefully tinker with features within the active site. Baker and colleagues have now developed what looks to be a huge leap forward in rational design. The authors use their approach to develop novel retro-aldol enzymes that can break a carbon-carbon bond in a non-natural substrate. They start by examining the active site of two structurally known retro-aldol enzymes. Because they were looking to design an enzyme that catalyzes a multi-step reaction, they first computationally modeled the different reaction intermediates and transition states. To improve the chances of recreating an active site that can stabilize a given transition state but also favorably interact with reaction intermediates and the other steps in the reaction pathway, the authors fine-sampled the degrees of freedom of the relevant functional groups to produce large ensembles of models for each of the key intermediate and transition states. These models were superimposed to produce several composite active site motifs capable of accommodating all reaction steps. The authors then used RosettaMatch to identify potential catalytic pockets from a library of protein scaffolds. After further optimization of the catalytic site and surrounding scaffold, the highest ranking models, based on binding energies and catalytic site geometry, were experimentally characterized. Of the 70 designs ultimately tested, 32 showed weak catalytic activity, with the most active designs having a modeled water molecule presumably involved in a proton shuffling step. The crystal structure of the best enzyme largely confirmed the modeled active site design. While this work showed that it is possible to design novel enzymatic activity, the best designs had activities that were only a fraction of those found in naturally-occurring enzymes, suggesting that additional features must be considered when optimizing the catalytic design.
