EMD-21021
MicroED structure of an oxindole product from modified trpB at 0.9A resolution
EMD-21021
Electron Crystallography0.9 Å
Deposition: 21/11/2019Map released: 11/12/2019
Last modified: 01/01/2020
Sample Organism:
Escherichia coli
Sample: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
Deposition Authors: Dick M, Sarai NS, Martynowycz MW, Gonen T, Arnold FH
Sample: (S)-2-amino-3-((S)-3-methyl-2-oxoindolin-3-yl)propanoic acid
Deposition Authors: Dick M, Sarai NS, Martynowycz MW, Gonen T, Arnold FH
Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation.
Dick M 
,
Sarai NS ,
Martynowycz MW ,
Gonen T ,
Arnold FH
(2019) J. Am. Chem. Soc. , 141 , 19817 - 19822
,
Sarai NS ,
Martynowycz MW ,
Gonen T ,
Arnold FH
(2019) J. Am. Chem. Soc. , 141 , 19817 - 19822
Abstract:
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.
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.