Engineered Biosynthesis of β-Alkyl Tryptophan Analogs

<a>Non-canonical 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 multi-step methods used to prepare them. Here we report a stereoselective biocatalytic synthesis of β-branched tryptophan analogs using an engineered variant of <i>Pyrococcus furiosus</i> tryptophan synthase (<i>Pf</i>TrpB), <i>Pf</i>TrpB^7E6. <i>Pf</i>TrpB^7E6 is the first biocatalyst to synthesize bulky β-branched tryptophan analogs in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of <i>Pf</i>TrpB^7E6 was explored through X-ray crystallography and UV-visible light spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. <i>Pf</i>TrpB^7E6 provides an operationally simple and environmentally benign platform for preparation of β-branched tryptophan building blocks.