
Chemistry ETDs
Publication Date
2-14-2014
Abstract
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.
Language
English
Keywords
hotdog, thioesterase, coenzyme A, kinetic
Document Type
Thesis
Degree Name
Chemistry
Level of Degree
Masters
Department Name
Department of Chemistry and Chemical Biology
First Advisor
Dunaway-Mariano, Debra
First Committee Member (Chair)
Mariano, Patrick
Second Committee Member
Melancon, Charles
Recommended Citation
Wu, Yajun. "determining the function of hotdog-fold thioesterases." (2014). https://digitalrepository.unm.edu/chem_etds/49