As the number of chemical reactions requiring biocatalysis is rapidly increasing in the research field of biofuel and pharmaceutical synthesis, enzyme engineering becomes one of important biotechnology topics. The efficiency of enzyme engineering relies on the protein space to explore. In order to expand the number of genetically encoded amino acids, we have previously demonstrated the concept that non-natural amino acids introduced in an enzyme active site can be used to modulate enzyme inhibition (1). However, the broad application of this strategy is limited by a challenge to experimentally explore a large number of candidate pairs of mutation site/non-natural amino acid. In order to overcome this issue, we combined the computational protein design with site-specific incorporation of a non-natural amino acid technique. As a model case, we chose murine dihydrofolate reductase (mDHFR) and its poor substrate folate. The computational protein design was performed to identify potential mutation sites and appropriate non-natural amino acids in order to increase the enzyme binding affinity to its substrate. Then, mDHFR mutants containing a non-natural amino acid were prepared and subjected to kinetic analysis. As expected, several mDHFR mutants exhibit significantly higher kinetic efficiencies compared to that of wild-type mDHFR. Our results successfully demonstrate that the combination of computational protein design and non-natural amino acid incorporation is an effective way to control enzyme catalytic properties.(1) Zheng, S. and Kwon, I., Controlling Enzyme Inhibition Using an Expanded Set of Genetically Encoded Amino Acids, Biotechnol. Bioeng., 2013, 110, 2361-2370.

Increasing the catalytic efficiency of biocatalyst via rational active site design using non-natural amino acid

247th National Meeting (2014)

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