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Enzyme design and engineering by implementation of non-canonical amino acids in protein scaffolds

Periodic Reporting for period 1 - THIAZOLIUMenzyme (Enzyme design and engineering by implementation of non-canonical amino acids in protein scaffolds)

Reporting period: 2019-09-15 to 2021-09-14

There is a growing demand in the chemical and pharmaceutical industry to replace traditional chemical catalysis with environmentally benign approaches for the synthesis of high-value compounds. Enzyme-mediated transformations, i.e. using enzymes as catalysts, generally offer sustainability in combination with high selectivity and catalytic activity. However, most naturally occurring enzymes do not meet the requirements of large-scale industrial processes and/or do not catalyse new-to-nature reactions that are of relevance to the chemical and pharmaceutical industry with sufficient efficiency. As a consequence, the design of enzymes and protein therapeutics with tailored, new-to-nature properties is a long-standing goal in enzymology and cell biology. Nature generally uses 20 amino acids as building blocks for protein synthesis. However, this portfolio limits the options for engineering proteins with ‘un-natural’ activities. Recent developments in the expansion of the genetic code have the potential to revolutionise the design of novel enzymes; by reprogramming the genetic code, we could convey novel functionality into proteins and extend their properties. THIAZOLIUMenzyme aimed at incorporating thiazolium amino acids into the active site of promiscuous and highly evolvable de novo enzymes for orchestrating organocatalytic transformations of clinical and industrial interest. Such reactions, conventionally mediated by non-enzymatic, small molecule N-heterocyclic carbene (NHC) catalysts require high temperature and catalyst loading. It was envisioned that an engineered enzyme with the ability to catalyse such chemistry could overcome the drawbacks of these abiological catalysts, serving as a ‘greener’ biocatalytic alternative, and also perform the desired reactions in cells for medicinal purposes.
THIAZOLIUMenzyme aimed at incorporating thiazolium amino acids into the active site of enzymes to orchestrate organocatalytic transformations conventionally mediated by small molecule N-heterocyclic carbenes (NHCs). The synthesis of corresponding new non-canonical amino acids (ncAAs) containing thiazolium moieties were successful. As a next step, I worked extensively to develop amino-acyl tRNA synthetases (aaRS) capable of selectively charging a suppressor tRNA with the ncAA and incorporating it into a protein active site. Unfortunately, efforts to develop such synthetases using directed evolution proved unsuccessful, and subsequent analysis of the tRNA synthetases in vitro failed to identify an orthogonal enzyme. On the basis of these results, the focus for the remainder of the project involved alternative enzyme engineering-based strategies with naturally-occurring thiamine dependent enzymes. However, demonstrating starting activity for the reactions of interest proved unsuccessful. Last, a review covering the field of enzyme engineering for therapeutic and medicinal enzymes, which are advanced goals of the MSCA proposal, is currently in preparation.
The learning of this project highlighted limitations in developing orthogonal tRNA synthetases for generating artificial enzymes bearing the ncAAs of interest. As a result, the envisioned biocatalytic transformations using the artificial enzymes could not be achieved. Nevertheless, the learnings from this endeavour could be transferred to future projects. It is antipated that future efforts will pave the way for development of general strategies for creating enzymes with unique properties and provide a tool-box for efficient, environmentally- friendly and bioorthogonal organocatalysed reactions. The generated artificial biocatalysts will have attractive applications in research, medicine and industry.
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