Fatty acid and polyketide synthesis enzymes: sequences, structures, and mechanisms
The fatty acid and polyketide synthesis enzymes were studied, and findings compose this dissertation. The thioester-active enzyme database (ThYme) was constructed, which provides almost all known sequences of fatty acid and polyketide synthesis enzymes classified and organized into families based on amino acid sequence similarity. ThYme aids in predicting active sites, catalytic residues, and mechanisms of individual sequences, as well as providing a standardized nomenclature. Detailed and timely reviews on thioesterases, ketoacyl reductases, hydroxyacyl dehydratases, enoyl reductases, and acyl carrier proteins, as well as their products and pathways, were also done.
Normal vibrational mode analysis showed that the dynamic behavior of acyl carrier protein structures was conserved within families. Also, the tertiary structures of three acyl carrier proteins were predicted.
A thioesterase family was statistically divided into subfamilies based on primary structure. This guided the choice of sequences for experimental characterization, to explore their substrate specificities and enzyme activity spaces.
The mechanism of a thioesterase enzyme was studied using quantum mechanics/ molecular mechanics metadynamics simulations. Its mechanism was confirmed, the electronic and atomic details of its transition state were revealed, its associated energy barrier was estimated, and new evidence was found suggesting a proton donating catalytic residue previously not considered.