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Advancing Promiscuity Characterization of ThDP-Dependent Enzymes for Carbon-Carbon Bond-Forming Catalysis
Advancing Promiscuity Characterization of ThDP-Dependent Enzymes for Carbon-Carbon Bond-Forming Catalysis
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20250211151017
- ISBN
- 9798382760377
- DDC
- 660
- 서명/저자
- Advancing Promiscuity Characterization of ThDP-Dependent Enzymes for Carbon-Carbon Bond-Forming Catalysis
- 발행사항
- [Sl] : Northwestern University, 2024
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2024
- 형태사항
- 135 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-11, Section: B.
- 주기사항
- Includes supplementary digital materials.
- 주기사항
- Advisor: Tyo, Keith E. J.;Broadbelt, Linda J.
- 학위논문주기
- Thesis (Ph.D.)--Northwestern University, 2024.
- 초록/해제
- 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.
- 일반주제명
- Chemical engineering
- 일반주제명
- Bioengineering
- 일반주제명
- Organic chemistry
- 키워드
- Enzyme screening
- 기타저자
- Northwestern University Chemical and Biological Engineering
- 기본자료저록
- Dissertations Abstracts International. 85-11B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■020 ▼a9798382760377
■035 ▼a(MiAaPQ)AAI30996085
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a660
■1001 ▼aHoang Dinh, Nhatruc Tracey.▼0(orcid)0000-0002-9829-9869
■24510▼aAdvancing Promiscuity Characterization of ThDP-Dependent Enzymes for Carbon-Carbon Bond-Forming Catalysis
■260 ▼a[Sl]▼bNorthwestern University▼c2024
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2024
■300 ▼a135 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-11, Section: B.
■500 ▼aIncludes supplementary digital materials.
■500 ▼aAdvisor: Tyo, Keith E. J.;Broadbelt, Linda J.
■5021 ▼aThesis (Ph.D.)--Northwestern University, 2024.
■520 ▼aCarbon-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.
■590 ▼aSchool code: 0163.
■650 4▼aChemical engineering
■650 4▼aBioengineering
■650 4▼aOrganic chemistry
■653 ▼aCarbon-carbon bond
■653 ▼aEnzyme promiscuity
■653 ▼aEnzyme screening
■653 ▼aThDP-dependent enzymes
■653 ▼aOrganic molecules
■690 ▼a0542
■690 ▼a0202
■690 ▼a0490
■71020▼aNorthwestern University▼bChemical and Biological Engineering.
■7730 ▼tDissertations Abstracts International▼g85-11B.
■790 ▼a0163
■791 ▼aPh.D.
■792 ▼a2024
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17160427▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
