Computational design of new biocatalysts for organic synthesis

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Enzymes are the most efficient catalysts known. They can increase the rate of a reaction by up to 17 orders of magnitude while displaying exquisite regio-, chemo- and stereoselectivity. Because of these advantages, many enzymes are being exploited as biocatalysts in numerous industrial syntheses. Our research program aims to develop new biocatalysts for organic synthesis using protein engineering and design. To do this, we will exploit computational protein design (CPD) algorithms. These algorithms predict amino acid sequences that are energetically compatible with the target structure while simultaneously allowing a novel property to be introduced into the target protein scaffold. The power of CPD results from its capability to perform virtual screening of sequence spaces astronomically larger (>10E80 sequences) than those that can be experimentally tested. We will develop biocatalysts for the direct asymmetric synthesis of D-amino acids. These compounds are important chiral building blocks in the synthesis of many pharmaceuticals, fine chemicals, agrochemicals, and food additives. However, most D-amino acids are either unavailable as commodity chemicals or are prohibitively expensive. This has created a need for more efficient and economical methods for the synthesis of D-amino acids to be developed. Our research program will address this need by engineering aminotransferases so that they can synthesize a wide range of D-amino acids with any desired side chain. The general strategy that will be employed to achieve our goals is: 1) computational design of focused combinatorial libraries, 2) DNA library construction using standard molecular biology techniques, 3) protein library screening using methods amenable to high-throughput approaches, and 4) characterization of positive hits. This last step will require production and purification of mutants, and detailed steady-state kinetic characterization using an HPLC-based assay. Outcomes of this research program will be the development of new biocatalysts for the direct asymmetric synthesis of D-amino acids. These results will contribute to the development of a more eco-efficient alternative to available chemical syntheses, and the application of green chemistry in Canada.