(307e) Substrate Channel Evolution and Circular Permutation of an Esterase for the Synthesis of Cilastatin
SUBSTRATE CHANNEL EVOLUTION AND CIRCULAR PERMUTATION OF AN ESTERASE FOR THE SYNTHESIS OF CILASTATIN
The esterase RhEst1 from Rhodococcus sp. ECU1013 has been reported for the enantioselective hydrolysis of ethyl (S)-(+)-2,2-dimethylcyclopropane carboxylate, producing the building block of cilastatin [1, 2]. Cilastatin can protect the kidneys against potential toxic effects from higher doses of imipenem. Imipenem and cilastatin have been used in combination as a 1:1 mixture, known as Tienam, which showed a broad spectrum activity and fewer side effects for the treatment of severe infections.
In this work , error-prone PCR and site-directed saturation mutagenesis were applied to RhEst1 for activity improvement, with the pH-indicator assay as a high-throughput screening method. RhEst1A147I/V148F/G254A, with mutations surrounding the substrate access channel, showed a 5-fold increase in its specific activity compared with the native enzyme, as well as a 4-fold increase in protein solubility. Combined with the determination of protein structures and computational analysis, this work shows that the amino acids around the substrate channel play a more important role in the activity evolution of RhEst1 than those in the active site. Mutations surrounding the entrance to the substrate channel allowed for an effective balance between improvement in the activity and a high level of enantioselectivity, which is a big challenge to improve the activity of RhEst1 towards ethyl 2,2-dimethylcyclopropane carboxylate. Circular permutation as a protein engineering approach was also applied to acquire functional variants of RhEst1. 98 CP mutants were constructed from three different sectors that we defined and the active variants mainly located on more flexible turn loops based on structure of RhEst1. The observed results suggest the relocation of protein terminus could be an effective method for protein engineering. The future work will be focused on the further evolution and structure analysis of the permutated RhEst1.
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