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PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
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Tryptophan Synthase: Biocatalyst Extraordinaire.

Ella Watkins-Dulaney1, Sabine Straathof2, Frances Arnold1,2

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Chembiochem : a European Journal of Chemical Biology
|July 18, 2020
PubMed
Summary

Tryptophan synthase (TrpS) is a key enzyme for synthesizing noncanonical amino acids (ncAAs). Directed evolution of the TrpB subunit has overcome limitations, expanding its utility in biocatalysis.

Keywords:
amino acidsbiocatalysisbiotransformationenzymeprotein engineering

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Area of Science:

  • Biochemistry
  • Enzymology
  • Synthetic Biology

Background:

  • Tryptophan synthase (TrpS) is crucial for l-tryptophan synthesis via a C-C bond-forming reaction.
  • Native TrpS complexes have limitations in engineering due to allosteric regulation and heterodimeric structure.
  • Noncanonical amino acids (ncAAs) offer diverse applications in synthetic and biological contexts.

Purpose of the Study:

  • To review the applications of TrpS in ncAA synthesis.
  • To highlight the advancements in engineering TrpS for broader substrate scope and enhanced properties.
  • To discuss the role of TrpS in biocatalytic cascades.

Main Methods:

  • Directed evolution of the TrpS β-subunit (TrpB).
  • Overcoming allosteric regulation by the TrpA subunit.
  • Engineering enzyme specificity for indole analogues.

Main Results:

  • TrpB was successfully evolved to be independent of TrpA allosteric regulation.
  • Significant expansion of TrpS substrate scope for ncAA synthesis.
  • Demonstrated utility of engineered TrpS in biocatalytic applications.

Conclusions:

  • Engineered TrpS, particularly TrpB, is a versatile biocatalyst for ncAA synthesis.
  • Directed evolution strategies have overcome key limitations of native TrpS.
  • TrpS holds great potential for future synthetic and biological applications.