The Evaluation of Synthetically Useful Aldolases

Duke University, Durham NC

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Abstract

DESCRIPTION (provided by applicant): The differing effects of antipodes of racemic pharmaceuticals have prompted the use of enantiomerically pure materials as therapeutic agents and have exacerbated the long-standing need for methodologies for enantiomerically pure synthesis. Since enzymes demonstrate remarkable selectivity in their transformations, they are key components in industrially feasible methods for synthesis of fine chemicals and pharmaceuticals. The pyruvate aldolases can be used as catalysts for stereocontrolled carbon-carbon bond formation in the synthesis of pharmaceutical intermediates. However, the rigid substrate specificity of this enzyme limits its utility in organic synthesis. We propose here a unique approach towards understanding the structural basis of enzyme catalysis and molecular recognition that combines synthetic organic chemistry, molecular biology, mechanistic enzymology, and X-ray crystallography. In toto, we aim to provide a new group of synthetically useful catalysts, develop methodologies for directed evolution, and extract detailed enzyme mechanistic information that will be interpreted in terms of enzyme structure. Specifically, we will: (1) Expand and/or shift the substrate specificity range of 2-keto-3-deoxy-6-phosphogluconafe (KDPG) aldolase to use both novel electrophiles and nucleophiles; (2) Demonstrate the synthetic utility of novel biocatalysts through concise environmentally benign syntheses of pharmaceutical intermediates; (3) Evaluate the catalytic behavior of mutated enzymes with a range of electrophilic and nucleophilic substrates; and (4) Determine the molecular structure of the mutated proteins through X-ray diffraction techniques. Together, these studies will significantly advance the goal of rational catalyst/biocatalyst design/redesign and enhance the synthetic utility of the pyruvate aldolases.