The bikaverin polyketide synthase (Pks4) synthesizes the small, anticancer molecule bikaverin. The C2-C7 first ring cyclization of bikaverin is directed by the product template (PT) domain of Pks4. To elucidate the molecular basis of cyclization in the fungal PKS, the Pks4 PT domain was cloned from Gibberella fujikuroi, expressed, and purified via Ni-IMAC chromatography and FPLC. Pks4 PT crystals diffracted to 3.1 Å. Further, site-directed mutagenesis was used to probe the mechanism of the Pks4 PT domain. The Pks4 PT crystal structure and mutagenesis will shed light on the cyclization mechanism and specificity of the fungal PKS. Additionally, a PT domain from Colletotrichum lagenarium (Pks1 PT) was expressed, purified, and crystallized. The Pks1 PT crystals diffracted to low resolution and are currently being refined. The two PT structures, combined with a PksA PT polyketide analogue co-crystal structure, will provide a starting point for rational bioengineering of the fungal PKS. In parallel, the starter unit:ACP transacylase (SAT) domains from the Pks4 and the aflatoxin PKS (PksA) were studied. SAT domains load starter units onto the ACP domains of the PKS. The SAT domains were expressed and purified via Ni-IMAC chromatography and FPLC. PksA SAT crystals diffracted to 2.1 Å and Pks4 SAT crystals diffracted to low resolution. The structure of the SAT domain will help determine the mechanism governing SAT substrate selectivity.
|Commitee:||Goulding, Celia, Poulos, Thomas|
|School:||University of California, Irvine|
|Department:||Chemistry - M.S.|
|School Location:||United States -- California|
|Source:||MAI 49/04M, Masters Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Chemical engineering|
|Keywords:||Crystallography, Product template, Starter unit:ACP transacylase|
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