Thioesterases are a superfamily of enzymes, which catalyze the cleavage of thioester bonds within a wide range of substrates, and are ubiquitously expressed in organisms ranging from prokaryotes through to humans. They have been classified into 25 families based on their sequence conservation, tertiary and quaternary structure, active site residues, and substrate specificity. They are also essential in a number of pathogenic bacteria, and therefore represent targets for drug design. This thesis characterizes the structural and functional elements governing thioesterase biology, including some novel mechanisms of thioesterase regulation. Moreover, new structural insights are established into a number of thioesterase families for which there was previously little information available. This study elucidates the structure of human ACOT12, as well as the structural basis for regulation by ADP and ATP. Prior to this, regulation was thought to be mediated through ligand-induced oligomerization of the thioesterase domains, with ATP-activated ACOT12 forming active dimers and tetramers whilst apo- and ADP-bound ACOT12 are monomeric and inactive. Using a range of biochemical and biophysical techniques, this study establishes that ACOT12 is a trimer, and the regulatory effects of ADP and ATP on the structure are mediated through two novel regulatory regions. In this thesis, the structure of the full length ACOT7, a homologue of ACOT12, associated with inflammation and overexpressed in the brain is presented. Interestingly, the structure of ACOT7 reveals a mechanism of domain rearrangement that has not been previously described in any thioesterase family, and may explain a number of interesting behaviors exerted by this enzyme. The overall quaternary structure is a hexamer of hotdog domains, arranged in a trimer-of-dimers. However, rather than an expected N-C N-C N-C arrangement, each double hotdog is positioned in an N-C C-N N-C configuration, which has a number of consequences for the type of domain interfaces that drive oligomerization. The structural and biophysical properties of two bacterial tesB thioesterases are presented, redefining aspects of the TE4 family. One family is shown to form an inactive enzyme complex, which appears to be conserved in a wide range of Mycobacteria. The other family forms an active tetramer of double hotdog dimers, which prior to this study, was annotated as forming a dimer. The tetramer of double hotdog dimers is shown to be conserved in a number of other prokaryotes. This study led to the discovery that tesB thioesterases may form an active tetramer mediated by a Glu residue, or an inactive dimer characterized by the mutation of the catalytic Asp residue to an Ala, as well as a number of defining features such as the presence of a ÃƒÂ¯�Ã‚°-helix at the active site. Finally the structure of a thioesterase from Legionella pneumophila revealed an unusual active site consisting of five residues. Significantly this active site has not been identified in any thioesterase previously and is conserved within members of the TE5 family. Overall this thesis redefines many aspects of the structural, functional, and biophysical aspects of a number of thioesterase families, providing novel and significant insights into the important thioesterase superfamily.
|Qualification||Doctor of Philosophy|
|Award date||01 Jun 2015|
|Place of Publication||Australia|
|Publication status||Published - 2015|