Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

We previously engineered the β-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 N1 of the substrate. Protecting N1 encouraged
evolution toward C-alkylation, which persisted when protection was
removed. Six generations of directed evolution resulted in TrpB Pfquat 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 γ-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 γ-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 Pfquat offers an efficient, sustainable, and
selective platform for the construction of diverse noncanonical amino
acids bearing all-carbon quaternary stereocenters.