Biocatalytic cascades involving LmrR-based artificial enzyme

A key challenge of integrating non-natural catalytic residues into proteins to design artificial enzymes for novel reactions lies in achieving in vivo reactivity due to issues such as metal cofactor toxicity and catalyst susceptibility to the biological milieu. Proposed solutions include compartmentalization strategies and incorporation of non-natural amino acids as catalytic moieties within biomolecular scaffolds.
To address this challenge of in vivo reactivity, this thesis is dedicated to the integration of specific unnatural amino acids. The results promise insights into the potential of hybrid enzymatic approaches to revolutionize biocatalysis for emerging applications.
This includes integrating para-amino-phenylalanine (pAF), an unnatural amino acid, into the LmrR protein. The modified protein functions as an artificial enzyme for cascade reactions, resulting in successful in vivo hydrazone formation. By coupling natural enzyme pathways with engineered LmrR-pAF, the study demonstrates intricate cascade catalysis, enabling biosynthesis of aromatic aldehydes.
Expanding the investigation, the research navigates complex reactions, exemplified by the Friedel-Crafts alkylation. Orchestrating a cascade involving artificial and natural enzymes, the study achieves controlled chemical product formation in vitro. While challenges remain for in vivo translation, the study identifies areas for refinement.
Furthermore, the study explores creating novel artificial enzymes by introducing non-canonical amino acids (N-Methyl Histidine and 3-Pyridyl Alanine) into the LmrR scaffold. Catalytic activity is successfully demonstrated in ester hydrolysis, with future trajectories including intricate chemical transformations.
In summary, this research pioneers hybrid enzyme systems, propelling the development of novel metabolic pathways in living cells.