Crystal Structure of Self-Spliced Group II Intron

摘要

Designing and creating nonnatural enzymes to catalyze synthetic reactions is a challenging and promising new frontier in the field of protein engineering and requires deep understanding of enzyme catalysis. The present study made use of a combination of computational design and directed evolution to create enzymes that will catalyze the Kemp elimination, an important synthetic reaction that does not have a naturally occurring enzyme catalyst and is physically restricted by high activation energy. The critical step in creating the enzyme is the choice of the catalytic mechanism and the design of the idealized active site (Fig. 1). Because the reaction requires proton transfer, the investigators chose two different bases: the carboxyl side chain of either glutamate or aspartate or the imidazole of histidine joined to either aspartate or glutamate (His-Asp or His-Glu dyad). The idealized active site was created by employing quantum mechanical transition-state calculations to position protein functional groups, specifically aromatic amino acid side chains that would provide maximum stability to the transition state while maintaining protein stability. Using the newly developed RosettaMatch hashing algorithm, protein backbone positions that would be capable of supporting these ideal active sites were screened. Eight designs showed promising activity in Kemp elimination assays. Characterization of the active sites of these eight designs showed the TIM barrel protein scaffold. To determine whether these active sites supported efficient catalysis, mutational analysis and high-resolution crystal structure studies were performed. Indeed, mutation of the catalytic bases either decreased or abolished the catalytic function of the enzyme. Directed evolution methods in vitro were also shown to improve stability and increase catalytic activity of the designed enzymes. The computational experimental design described in the article in combination with directed evolution studies for designing novel enzymes provides a new strategy for creating novel and efficient enzymes for many synthetic reactions for which naturally occurring enzymes do not exist.