The use of covalent modification andgenetic code expansion to createorganocatalytic artificial enzymes

Cyclic secondary amines can be used to perform enamine and iminium ion catalysis to modify carbonyl compounds through a broad range of chemical reactions. To utilise these compounds in a chemical biology context requires the catalysis to work in an aqueous and biocompatible environment. To accomplish this, the catalysts can be placed into proteins generating new hybrid organocatalytic artificial enzymes. This thesis concerns two strategies to generate these artificial enzymes and the subsequent characterisation of their activity. Firstly, penicillin binding proteins are covalently modified with carbapenems that carry secondary amine pyrrolidines in their structure. Both the carbapenems alone and the protein-carbapenem hybrids were able to afford a Michael-nitro addition reaction under biocompatible reaction conditions. The second strategy concerns the direct incorporation of secondary amine unnatural amino acids into a protein backbone using genetic code expansion technology. Three unnatural amino acids are incorporated at four different positions in the multidrug binding protein LmrR. The resulting proteins are tested for their activity to perform selective reduction of α,β- unsaturated aldehydes using benzyl-nicotinamide as a hydride source. The catalytic amino acid generating the highest conversion is then incorporated into the natural oxidoreductase dihydrofolate reductase from E. coli at three different positions. The EcDHFR variants are then tested for their activity in the hydride transfer reaction, using NADPH as the hydride source. Two highly active enzymes LmrR Phe93DPK and EcDHFR Ala7DPK are identified and further investigated for their enzyme kinetic character and substrate scope. EcDHFR Ala7DPK is then coupled to a natural enzyme system allowing for the co-factor to be recycled in situ. Mechanistic studies and kinetic isotope effects are performed that strongly suggest an iminium ion mechanism in the hydride transfer reaction. Genetic code expansion relies heavily on orthogonal tRNA synthetases. In this work, pyrrolysine tRNA synthetase form M. bakeri is used as the pivotal in vivo enzyme to incorporate the cyclic secondary amine unnatural amino acids. Although the catalytic C-terminus has been extensively explored, the tRNA binding N-terminus has been less so. Mutations in the N-terminus were performed to determine if they can increase unnatural amino acid incorporation efficiency, however the opposite was found, in contrast to its homolog from M. mazei. Five MbPylRS variants, eight unnatural amino acids and two different culture temperatures are explored.