TOWARDS THE STRUCTURAL DETERMINATION OF ENXKS0

Polyketides are a large class of bioactive secondary metabolites that are widely used in the pharmaceutical industry due to their diverse activities. They are produced by large enzyme complexes called polyketide synthases (PKS) which are studied as platforms for generation of bioactive compound libraries for drug development. The polyketide Enacyloxin IIa from Burkholderia ambifaria has antibiotic activity towards a historically antibiotic resistant bacterium B. multivorans that is known to cause deadly infections in cystic fibrosis patients. EnacyloxinIIa’s unusual PKS has several unique domains including a novel type of ketosynthase (KS) termed KS⁰. A typical KS extends the growing polyketide chain by two carbons via a decarboxylative Claisen condensation, but the KS⁰ does not display this activity. The KS0 is essential for product formation, though its function is unknown. Additionally, KS⁰ has high sequence identity with canonical KSs, so the basis for any unique activity is unclear. The hypothesis of this study is that KS⁰’s function is to transfer the growing polyketide chain from an upstream acyl carrier protein domain to a downstream peptide carrier protein domain. In order to test this hypothesis, our goal was to solve the crystal structure of the enzyme in order to gain structural insight into its function. In order to increase the probability of crystallization, five 6xHis-tagged constructs with varying C- and N-terminal linker regions were generated. These constructs were expressed in E. coli and purified using nickel affinity, anion exchange, and size exclusion chromatography. The protein was then screened against over one thousand unique crystallization conditions. The initial crystal hits were then optimized by adjusting parameters such as protein concentration, precipitant concentration, pH, and small molecule additives. The crystals were also improved through microseeding. The largest crystals were observed in 0.1 M MES pH 8.1, 0.1 M MgCl₂, 0.36 M NDSB-256, and 30% PEG 400. The crystals were flash frozen in liquid nitrogen and subjected to x-ray diffraction analysis at the Advanced Light Source synchrotron facility. The data were processed and scaled to a resolution of 3.05 Å. Currently, the resolution is not sufficient to solve the structure using molecular replacement. A homology model of KS⁰ was created using the i-Tasser server. The model predicts a high structural homology with typical KS domains. Through comparison with a KS from a similar PKS, it was observed that KS⁰ has an alanine residue instead of a conserved histidine that is important for decarboxylation. This could explain why KS⁰ does not extend the polyketide chain. Although we have thus far been unable to solve the structure, the crystals are still being optimized. Additionally, we plan to utilize surface entropy reduction mutants to improve the crystal packing and increase the resolution.