자료유형  

 학위논문

Control Number  

0016932838

International Standard Book Number  

9798379851552

Dewey Decimal Classification Number  

551.5

Main Entry-Personal Name  

Boram, Trevor.

Publication, Distribution, etc. (Imprint  

[S.l.] : Purdue University., 2022

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2022

Physical Description  

1 online resource(164 p.)

General Note  

Source: Dissertations Abstracts International, Volume: 85-01, Section: B.

General Note  

Advisor: Lohman, Jeremy R.

Dissertation Note  

Thesis (Ph.D.)--Purdue University, 2022.

Restrictions on Access Note  

This item must not be sold to any third party vendors.

Summary, Etc.  

요약Biosynthesis of fatty acids and specialized metabolites, such as polyketides, is dependent on the C-C bond forming enzymatic activity of carboxylases and ketosynthases (KS). Carboxylases and KS perform complex carbon-carbon bond forming reactions via a ping-pong mechanism; the catalytic interactions of which are still unclear. The KS reaction involves the Claisen condensation of an acylated enzyme with a malonyl-thioester, driven forward by the energy of the malonyl-thioester decarboxylation. Similarly, the carboxylase proceeds via a carboxyl-biotin-enzyme intermediate, and a subsequent C-C bond forming reaction. Engineering the substrate specificity of these enzyme involved in producing polyketides is sought after for the purpose of producing novel, derivative polyketides. These derivative polyketides may have serve as effective new antibiotics, of which discovery has waned. Unfortunately, incomplete understanding of protein-protein interactions, conformational changes, and substrate orientation in catalysis leads to not well informed engineering attempts. A challenge in deducing the catalytic details of enzymes acting on malonyl-thioesters in general is the hyper-reactivity of their βketoacid and thioester substrates, which are prone to hydrolysis and decarboxylation. Many structures of malonyl-CoA bound enzymes feature hydrolysis of the thioester, preventing determination of enzyme:substrate interactions in structure-function studies. To work around this problem of innate reactivity, groups have synthesized a variety of acyl-thioester analogs for probing the details of enzyme catalysis with mixed success. The success of these enzyme:analog mechanistic studies appears to hinge upon the similarity of the analog to the natural substrate. Here, we demonstrate the synthesis of near-natural, acyl-thioester analogs, featuring single atom substitutions. Using a novel UV-vis assay, we have determined Ki values of our analogs with paradigmatic KSs E. coli FabH. These Kivalues are marginally higher than the substrate Km values, suggesting the KSs bind the analogs as they would natural substrates. Using this information, we have conducted preliminary X-ray crystallography experiments to determine the carboxylase:analog and KS:analog catalytic interactions, which will allow for new insight into debated C-C bond forming catalytic details. The information presented in this thesis and additional studies on protein-protein interactions can be leveraged into informed engineering studies of PKS enzymes.

Subject Added Entry-Topical Term  

Hydrocarbons.

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Success.

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Biosynthesis.

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Fatty acids.

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Chemistry.

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Chromatography.

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Research & development--R&D.

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Mass spectrometry.

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Oxidation.

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Herbicides.

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Antibiotics.

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Cloning.

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Adenosine triphosphate.

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Carbon dioxide.

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Chemical bonds.

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Engineering.

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Catalysis.

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Natural products.

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Enzymes.

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Polypeptides.

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Crystallography.

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Agricultural chemistry.

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Organic chemistry.

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Pharmaceutical sciences.

Added Entry-Corporate Name  

Purdue University.

Host Item Entry  

Dissertations Abstracts International. 85-01B.

Host Item Entry  

Dissertation Abstract International

Electronic Location and Access  

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Control Number  

joongbu:644012