Chemistry ETDs


Yajun Wu

Publication Date



The enzymes that hydrolyze thioesters to the free acids and free thiols are called thioesterases. Based on the different folding strategy, most thioesterases are classified into two superfamiles: the hotdog-fold enzyme superfamily or alpha/beta-fold hydrolase enzyme superfamily. Hotdog-fold thioesterases share high degree similarity in structure which resembles a hotdog'. YigI is one of the last two uncharacterized hotdog-fold thioesterases from E.coli. To study the function of YigI, substrate specificity profile was determined by thioesterase activity assay. YigI possesses a preference for long chain acyl-CoA over shorter chain acyl-CoA. The highest activity was found in catalysis of myristoyl-CoA. By alignment with the closest homolog, the uncharacterized tetrameric type AB thioesterase from Shewanella oneidensis, the important catalytic residues of YigI were predicted as following: Asp69, Gln56 and His60. Enzyme activity decreased after the site-direct mutagenesis of those residues. Cytoplasmic acetyl-coenzyme A hydrolase (CACH), also known as acyl-CoA thioesterase12 (Acot12), plays important cellular roles in mammalian fatty acid metabolism through the hydrolysis of acyl-CoA thioesters. Unlike YigI, besides two hotdog domains, CACH contains a steroidogenic acute regulatory (START) domain. The START domain is known as a lipid transporter. Thus, the function and regulation of CACH is f special interest. Chapter three of this thesis focuses on the function of the tandem hotdog-fold domains of CACH. The double hotdog-fold unit of CACH was expressed in E. coli and purified. Surprisingly, it was shown to display highest activity towards isobutyryl-CoA rather than acetyl-CoA.




hotdog, thioesterase, coenzyme A, kinetic

Document Type


Degree Name


Level of Degree


Department Name

Department of Chemistry and Chemical Biology

First Advisor

Dunaway-Mariano, Debra

First Committee Member (Chair)

Mariano, Patrick

Second Committee Member

Melancon, Charles