Directed Evolution of an Allosteric Tryptophan Synthase to Create a Platform for Synthesis of Noncanonical Amino Acids

Tryptophan and its derivatives are important natural products and have many biochemical and synthetic applications. However, the more elaborate these derivatives are, the more complex the synthesis becomes. In this chapter, we summarize the development of an engineered enzymatic platform for synthesis of diverse tryptophan analogs. This endeavor utilizes the tryptophan synthase (TrpS) enzyme, an α2β2 heterodimeric protein complex that catalyzes the last two steps in the biosynthetic pathway of tryptophan. Although the synthetically useful reaction (indole + Ser = Trp) takes place in the β-subunit (TrpB), the exquisite allosteric regulation of this enzyme impedes the use of isolated TrpB due to its dramatically decreased activity in the absence of the α-subunit (TrpA). This chapter discusses our efforts to engineer TrpB to serve as a general platform for the synthesis of noncanonical amino acids. We used directed evolution to enhance the activity of TrpB from Pyrococcus furiosus (PfTrpB), so that it can act as a stand-alone biocatalyst. Remarkably, we found that mutational activation mimics the allosteric activation induced by binding of TrpA. Toward our goal of expanding the substrate scope of this reaction, we activated other homologs with the same mutations discovered for PfTrpB. We found improved catalysts for the synthesis of 5-substituted tryptophans, an important biological motif. Finally, we performed directed evolution of TrpB for synthesis of β-branched amino acids, a group of products whose chemical syntheses are particularly challenging. [email protected]