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TitleTailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation.
Journal, issue, pagesJ Am Chem Soc, Vol. 141, Issue 50, Page 19817-19822, Year 2019
Publish dateDec 18, 2019
AuthorsMarkus Dick / Nicholas S Sarai / Michael W Martynowycz / Tamir Gonen / Frances H Arnold /
PubMed AbstractWe 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 ...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 N of the substrate. Protecting N encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB 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 culture, TrpB offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.
External linksJ Am Chem Soc / PubMed:31747522 / PubMed Central
MethodsEM (electron crystallography)
Resolution0.9 Å
Structure data

EMDB-21021:
MicroED structure of an oxindole product from modified trpB at 0.9A resolution
Method: EM (electron crystallography) / Resolution: 0.9 Å

EMDB-21022:
MicroED structure of a ketone product from modified trpB at 0.9A resolution
Method: EM (electron crystallography) / Resolution: 0.9 Å

Source
  • Escherichia coli (E. coli)

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