Research Projects

Rationale: Biological systems strongly favor one enantiomer over the other, as seen with D-sugars and L-amino acids, a preference driven by highly enantiospecific enzymes.However, the importance of the opposite enantiomers—L-sugars and D-amino acids—is increasingly being recognized in natural systems. They are also valuable as starting materials for the enzymatic or organic synthesis of pharmacologically active molecules. Due to the natural scarcity of L-sugars and D-amino acids, these processes can be costly. Enzymatic synthesis of D-amino acids and their derivatives could offer a more efficient alternative. Enzymes that act on L-amino acids could be engineered to recognize D-amino acids. While understanding on how enzymes achieve enantiospecificity is available, a seamless approach to switch enantiospecificity in enzymes is missing.. Hypothesis/Model: The active sites of enzymes with opposite enantiospecificity are typically related by mirror or plane symmetry, offering the potential for switching enantiospecificity by directly mutating the active site residues. However, achieving this switch through rational mutagenesis is challenging, as it often fails to account for factors like active site dynamics and the role of water molecules in catalysis. A streamlined approach using directed evolution will be used to switch enantiospecificity in two different enzyme systems representing different models of enantiospecificity. Objectives: 1) Switching the enantiospecificity in Escherichia coli L-tartarate dehydratase: a case study of an enzyme enantiospecifity with enantiomer superposition model 2) Switching the enantiospecificity in Bacillus subtilis L-glutamate dehydrogenases: a case study of the four-contact point based enantiospecificity 3) Evolving enantiospecific enzymes using racemases as templates Experiments: Directed evolution will be employed to switch the enantiospecificity of enzymes L-tartarate dehydratase and L-glutamate dehydrogenase. Mutant E. coli and Bacillus strains will be used for selection. The variants with switched enantiospecificity will be subjected to biochemical, kinetic and structural studies. For the third objective, a bioinformatic analysis—including phylogenetic tree construction and evolutionary conservation of domains—will be performed on various racemases. Specific cases will then be selected for experimental validation. Significance: Most drugs used for pharmacological intervention are highly stereospecific, and enzyme-mediated catalysis offers an efficient way to produce these molecules. Unlike organic synthesis, enzyme catalysis can yield highly stereospecific compounds of interest. The proposed experimental model and strategy will provide valuable insights into how enzymes achieve stereospecificity. The results could serve as a general approach for switching enantiospecificity in enzymes, with potential applications in synthetic biology and the pharmaceutical industry.