Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Molecular, Cellular and Developmental Biology
Basil J. Nikolau
Fatty acids are an integral part of the lipid membrane of bacteria and plants, and the enzyme that initiates fatty acid synthesis, β-ketoacyl-ACP Synthase III (KASIII), is one of the most diverse enzymes found in nature. KASIII exhibits vast diversity in its substrate specificity. By virtue of its substrate specificity, KASIII determines the fatty acid profile of the organism by dictating the nature of the ω-end of the fatty acids. For example, KASIIIs from many bacteria (e.g. Escherichia coli) accept straight chain acyl-CoAs resulting in production of straight chain fatty acids and are, therefore, narrow in substrate specificity. In contrast, KASIII from other bacteria (e.g. Staphylococcus aureus) accept both straight and branched chain acyl-CoAs resulting in production of both straight and branched chain fatty acids and thefore, exhibit broad substrate specificity. Despite the availability of KASIII crystal structures from a dozen sources, the molecular basis for its substrate diversity remains unclear. Additionally, the enzymatic activity of KASIII enzymes has not been explored with substrates other than straight or branched chain acyl-CoAs. However, occurrence of omega-cyclic and omega-phenylic fatty acids in certain bacteria and plants suggests that KASIII in such organisms may be capable of utilizing atypical substrates such as cyclic acyl-CoAs or aromatic acyl-CoAs.
In this dissertation, we have experimentally established the essentiality of three residues in determining KASIII substrate binding and catalysis. We have also characterized twelve putative KASIII enzymes from diverse bacterial sources and have identified six functional KASIIIs from this set. Additionally, based on phylogenetic and functional comparisons of all the characterized KASIIIs, we have developed a structure-function correlation that can be used for predicting KASIII functionality. Finally, we identified three novel KASIII enzymes that can utilize atypical substrates, including hydroxylated, aromatic and unsaturated acyl-CoAs. We subsequently demonstrated the in vivo production of novel omega-1-hydroxy-branched fatty acids using a novel KASIII. The knowledge gained from this study has enhanced our understanding of the KASIII structure-function relationship and has paved the way for developing fatty acids with novel chemical functionalities at the ω-end using KASIIIs with diverse specifcities.
Garg, Shivani, "Exploring and understanding the substrate diversity of b-ketoacyl-ACP synthase III enzymes" (2013). Graduate Theses and Dissertations. 16085.