Purdue University Graduate School
Trevor thesis FINAL resubmission.pdf (6.25 MB)


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posted on 2022-04-28, 23:20 authored by Trevor J. BoramTrevor J. Boram


Biosynthesis 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 β-ketoacid 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 Ki values 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.


NIGMS 1R01GM140290-01


Degree Type

  • Doctor of Philosophy


  • Biochemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Jeremy R. Lohman

Additional Committee Member 2

Frederick S. Gimble

Additional Committee Member 3

Mark C. Hall

Additional Committee Member 4

Carol B. Post