Document Type



Doctor of Philosophy (PhD)



First Advisor's Name

Xiaotang Wang

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Yuan Liu

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Barry Philip Rosen

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Manuel Alejandro Barbieri

Fourth Advisor's Committee Title

Committee member


Chloroperoxidase, crystallography, enantioselective, epoxidation, mutagenesis, mechanism

Date of Defense



The chloroperoxidase secreted from Caldariomyces fumago catalyzes broad spectrum of reactions. The crystallography combined with X-ray diffraction analysis was conducted to reveal recombinant CPO expressed in a modified Aspergillus niger system. Our results indicated that despite functional similarities with wild type CPO, recombinant CPO is over glycosylated with more mannose. Besides, ten iodide ion binding sites were identified in rCPO and six of them were found to be well superimposed on previously reported structure of the wild type CPO. Therefore, recombinant CPO shares almost the same structure with wild type CPO, and the Aspergillus niger is a potential system for heterologous heme peroxidase expression.

The most striking feature of C. fumago chloroperoxidase is its ability to efficiently catalyze asymmetric epoxidations of alkenes with excellent enantioselectivity. To elucidate the structural basis for the mechanism of CPO-catalyzed enantioselective epoxidation of selected olefins, the structures of the CPO and its olefin complex were studied by both crystallographic and computational techniques. Molecular docking studies demonstrated that ethyl 3-methylbut-3-enoate forms a complex with CPO at a relatively flexible channel through hydrogen bond interactions with Ala 75, coupled with other Pi-interactions between Phe186 and the carbon-carbon double bond of the substrate, ensuring its enantioselective transformation. CPO mutants of C29H, F186A and F103A/186A were expressed in A. niger and results of catalytic assays dwarfed the effects of thiolate-ligand, while confirming the roles of Phe103 and Phe186 in enzyme-substrate interactions and catalytic activities. The prokaryotic system was applied to express mutant CPO-C29H. Our work demonstrated that the heme binding of C29H is improved with the assistance of ChuA gene, but C29H expressed in E. coli cells is inactive in either chlorination or peroxidation, indicating the requirement of post-translational modifications for mutant CPO.





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Biochemistry Commons


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