Engineering the Tryptophan Synthase β-Subunit for Synthesis of Noncanonical Amino Acids

Author: Watkins-Dulaney, Ella Jenná

Year: 2022

Degree: Dissertation (Ph.D.)

Advisor: Arnold, Frances Hamilton

Committee Members: Shapiro, Mikhail G.; Ismagilov, Rustem F.; Tirrell, David A.; Buller, Andrew R.; Arnold, Frances Hamilton

Option: Bioengineering

DOI: 10.7907/yekm-y267

Abstract

The tryptophan synthase β-subunit (TrpB) naturally catalyzes a pyridoxal phosphate cofactor-mediated β-substitution reaction between indole and serine to form L-tryptophan. Almost half a century ago, it was realized that TrpB could accept nucleophiles other than indole to synthesize noncanonical amino acids (ncAAs), which are highly useful small-molecule building blocks that are found in many bioactive molecules. Since then, TrpB has been applied to synthesize a wide range of ncAAs. This thesis details the engineering of TrpB for synthesis of new and useful ncAAs and the application of TrpB as a model to study the principles that govern intra-protein interactions. Chapter I chronicles the history of tryptophan synthase, provides useful information about the enzyme’s catalytic cycle, and describes how TrpB has been used to synthesize ncAAs in works preceding this thesis. Chapter II describes the evolution, application, and characterization of TrpB for the synthesis of a blue, fluorescent noncanonical amino acid β-(1-azulenyl)-L-alanine (AzAla). Chapter III details the engineering and mechanistic characterization of TrpB to asymmetrically catalyze C–C bond formation with an entirely new class of nucleophile: ketones. Chapter IV describes the in vivo continuous evolution of TrpB which resulted in sequence-diverse TrpB orthologs that have been adapted to function at lower temperatures and display a range of substrate-selectivity profiles. Chapter V describes the development of a deep mutational scanning experiment of combinatorial site-saturation mutagenesis (SSM) libraries for generating a large dataset that maps enzyme sequence to function for the purpose of studying epistasis with machine learning. Overall, the work presented in this thesis expands the repertoire of ncAAs that can be synthesized by TrpB and demonstrates unique applications of TrpB as a model enzyme for continuous in vivo directed evolution and for generating a dataset that will be useful to the protein machine learning community.

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