Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

We previously engineered the beta-subunit of tryptophan synthase (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds on N-1 of the substrate. Protecting N-1 encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pf(quat) with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereocenter at the gamma-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the gamma-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pf(quat) offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.