자료유형  

 학위논문

Control Number  

0017160427

International Standard Book Number  

9798382760377

Dewey Decimal Classification Number  

660

Main Entry-Personal Name  

Hoang Dinh, Nhatruc Tracey.

Publication, Distribution, etc. (Imprint  

[S.l.] : Northwestern University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

135 p.

General Note  

Source: Dissertations Abstracts International, Volume: 85-11, Section: B.

General Note  

Includes supplementary digital materials.

General Note  

Advisor: Tyo, Keith E. J.;Broadbelt, Linda J.

Dissertation Note  

Thesis (Ph.D.)--Northwestern University, 2024.

Summary, Etc.  

요약Carbon-carbon bonds make up the backbone of all organic molecules, and their formation is a key reaction step in the synthesis of large, complex compounds. Traditional chemical synthesis routes toward C-C bond formation often require multiple steps for protection and deprotection of non-reacting functional groups and rely on highly reactive metal catalysts in acidic or basic conditions. As a highly desirable alternative, biocatalysts offer highly specific, single step carboligation at mild operating conditions. A variety of enzymes catalyzing carboligation have been discovered; however, their implementation in organic synthesis has been constrained by limited characterization of their substrate scope. Thiamine diphosphate (ThDP)-dependent enzymes are one such group that exhibit non-native activity, also known as promiscuity, for asymmetric carboligation between a variety of carbonyls. Despite the large number of ThDP-dependent enzymes that have been identified for carboligation, the range of substrate promiscuity characterized for each enzyme is quite limited. This dissertation seeks to broaden the scope of known promiscuity profiles of ThDP-dependent enzymes that carry out carboligation. In doing so, we aim to harness their versatility for new-to-nature reaction discovery and novel product formation and to understand the implications of their promiscuous activity within the cellular context.We first demonstrate a platform to rapidly characterize enzyme substrate promiscuity for bimolecular carboligation reactions. This assay screens hundreds of diverse pairs of α-keto acid substrates by multiplexing reactions and relying on high-resolution liquid chromatography mass spectrometry (LC-MS) for multiple product detection. We demonstrate analytical strategies necessary to refine complex spectrometry data and to generate enzyme activity data for 17 ThDP-dependent enzymes. In total, we present a database of 3376 positive and negative enzyme-substrate paired carboligation reactions, the largest promiscuity database of ThDP-dependent enzymes to date. The utility of this dataset for developing predictive promiscuity models was demonstrated in a preliminary activity classifier. Lastly, we identified cellular burden during expression of ThDP-dependent enzymes and explored the contribution of its promiscuous activity to cellular toxicity.In all, this dissertation broadens our understanding of the promiscuous capability inherent in ThDP-dependent enzymes. It establishes a basis for further characterization of their promiscuity for other types of substrates and provides valuable data necessary for developing predictive enzyme promiscuity models. In profiling carboligases for new reactions, we also aim to advance the incorporation of ThDP-dependent enzymes as versatile biocatalysts in the synthesis of complex molecules.

Subject Added Entry-Topical Term  

Chemical engineering.

Subject Added Entry-Topical Term  

Bioengineering.

Subject Added Entry-Topical Term  

Organic chemistry.

Index Term-Uncontrolled  

Carbon-carbon bond

Index Term-Uncontrolled  

Enzyme promiscuity

Index Term-Uncontrolled  

Enzyme screening

Index Term-Uncontrolled  

ThDP-dependent enzymes

Index Term-Uncontrolled  

Organic molecules

Added Entry-Corporate Name  

Northwestern University Chemical and Biological Engineering

Host Item Entry  

Dissertations Abstracts International. 85-11B.

Electronic Location and Access  

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Control Number  

joongbu:657375