Engineering amide bond ligases (ABLs) for industrial pharmaceutical production'

The amide bond is one of the most significant in pharmaceutical chemistry, featuring in a host of important everyday pharmaceuticals for the treatment of ulcers, high cholesterol and pathogenic infections by bacteria and viruses. It is vital therefore that there exist atom efficient and sustainable green chemical methods for the synthesis of pharmaceutical amides. However, industrial synthetic methods for the preparation of amides suffer from the use of complex or hazardous reagents to accomplish their chemistry and generate a large amount of waste. Because of this lack of efficiency, industrial synthetic chemists are increasingly turning towards 'biocatalysis' or 'Industrial Biotechnology' as the preferred method of synthesising molecules for pharmaceutical production. Biocatalysts, such as enzyme or microbes, typically achieve the synthesis of chemical bonds with excellent atom efficiency and selectivity, and Nature is also expert at synthesising amide bonds, which are the major bonds that hold the structure of proteins together. Until now however, biocatalysts for the formation of amide bonds have received little attention for industrial application, even though such enzyme reactions feature at the top of the list for many chemists looking for biocatalytic solutions to synthetic problems. This is because biocatalytic methods for amide bond formation in Nature, while efficient, are often complex, and difficult to apply out of their natural context. A recently discovered group of enzymes, which we have called amide bond ligases (ABLs), offers new and unexplored promise for biocatalytic amide bond formation, as their reaction chemistry is comparatively simple, and also because the kind of amide bonds that they form, are much more closely related to molecules of real pharmaceutical interest than has previously been the case. In this project, which is a collaboration between biochemists and synthetic chemists at York, and in association with GSK, we propose to thoroughly investigate the synthetic potential of the new ABL enzymes. First we will define the potential and limitations of the natural enzymes using a mixture of synthetic chemistry and biocatalysis. We will then solve the as-yet-undetermined structures of the ABL enzymes using X-ray crystallography and use rational enzyme redesign to expand the potential of the enzymes to catalyse the synthesis of a much wider range of pharmaceutically relevant molecules. We will also use contemporary protein evolution techniques to adapt the enzymes to act on alternative substrates that are of interest to industrial collaborators. This will involve the design and application of new high-throughput screens for amide bond formation to allow us to perform rapid improvement of the enzymes, and also apply new techniques in enzyme cofactor recycling to all