Engineered biosynthesis of β‐alkyl tryptophan analogs

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β‐branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of β‐branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB^(7E6). PfTrpB^(7E6) is the first biocatalyst to synthesize bulky β‐branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB^(7E6) was explored through X‐ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active‐site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB^(7E6) provides an operationally simple and environmentally benign platform for the preparation of β‐branched tryptophan building blocks