| Generation of small designer protein catalysts with desired reaction specificity is a challenging task. A new and very promising approach is the rational discovery of new promiscuous reactions in existing enzymes, and exploiting this promiscuity to create tailor-made biocatalysts. The goal of this proposal is to develop small designer enzymes for non-natural carbonyl transformation reactions on the basis of the b-a-b scaffold through directed evolution, rational engineering, genome mining, and chemical synthesis. The simple b-a-b scaffold, the building block of tautomerase superfamily proteins, has several features that make it an attractive candidate for such design. First, the tautomerase superfamily members have an inherent functional promiscuity in their catalytic mechanisms and substrate specificities, which stems in part from their ability to bind and polarize a carboxylate or carbonyl group. This feature represents an essential catalytic step in many carbonyl transformation reactions. Second, they use a catalytic amino-terminal proline (the only natural amino acid with a secondary amine group), which provides a unique chemical functionality at the N-terminus. The secondary amine can react with carbonyl compounds to form imine intermediates or enamines that facilitate many carbonyl transformation reactions. Third, the amino acid sequences that make up the scaffold are relatively short (~60 residues), and are therefore amenable to chemical synthesis. This allows for the introduction of noncoded amino acids, which expands further the novel catalysts that could potentially be generated from this basic scaffolding. This proposal will exploit this potential for promiscuity of the b-a-b scaffold for the creation of a new family of designer enzymes for non-natural aldol, alkylation, Michael addition, and other important enantioselective carbon-carbon bond-forming reactions. The resulting enzymes will have potential as biotechnologically useful catalysts for chemical and pharmaceutical synthesis and represent unique model systems to study imine- and enamine-based enzyme catalysis utilizing a proline or proline-like nucleophile. |
