Structural Basis for Chloroperoxidase Catalyzed Enantioselective Epoxidations and Mechanisms of Selected Anticancer Drug Induced Apoptosis

Authors

Yongjian Guo, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

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

Stanislaw F. Wnuk

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Manuel Alejandro Barbieri

Fourth Advisor's Committee Title

Committee member

Keywords

biotechnology, cell biology, medicinal chemistry and pharmaceutics, pharmacology

Date of Defense

11-14-2019

Abstract

Chloroperoxidase (CPO), a member of the heme peroxidase family, has diverse catalytic activities toward a broad range of substrates. In addition to catalyzing halogenation reactions involved in the biosynthesis of halogen-containing compounds, CPO also catalyzes reactions typical of traditional heme peroxidases, catalases, and cytochrome P450 enzymes. Despite the powerful and versatile catalytic activity of CPO, its applications have been thwarted by the difficulty in regenerating the active enzyme and substrate (peroxide) induced protein inactivation. To overcome these shorting comings of the protein, we investigate the fabrication and characterization of chloroperoxidase (CPO) and glucose oxidase (GOx) on the surface of MGO. The performance of the immobilized CPO was considerably enhanced by coupling with GOx that provided the required H₂O₂ in situ through glucose oxidation. The activity of MGO-GOx-CPO (96.6%) towards the decolorization of orange G was much superior to that of MGO-GOx+MGO-CPO (86.2%), probably as a result of reduced mass transfer resistance between CPO and H₂O₂ generated from GOx molecules. MGO-GOx-CPO can be conveniently reused for its ease of recovery in the presence of an external magnetic field, with ∼38.5% activity remained after 6 cycles of applications. The work detailed in Chapter 2 of this dissertation demonstrates the feasibility of co-immobilizing CPO and GOx onto MGO and the potential of MGO-GOx-CPO in environmental applications.The versatility of CPO has long been attributed to the unique structural components of the protein environment that constitute the heme active site, particularly the identity of the axial heme ligand and amino acid residues distal to the heme iron. The heme active site structure has also been thought to be responsible for the enantioselectivity of CPO. Chapter 3 of my dissertation investigates the structural factors that contribute to CPO’s stereoselectivity in catalyzing epoxidation reactions. The characterization of CPO-substrate complex will be carried out using UV-Vis spectrophotometry and nuclear magnetic resonance (NMR) spectroscopy. We will focus on understanding the mechanism of CPO-catalyzed enantioselective epoxidation and structure-activity relationship of CPO.

Non-small-cell lung carcinoma (NSCLC) continues to be a vital human healthcare problem worldwide for its high incidence and consequent mortality rate. In this study, we investigated the anticancer effect of LZ-101, a novel derivative of danofloxacin, against non-small-cell lung cancer and the underlying mechanisms. In vitro, LZ-101 inhibited the viability of A549 human non-small cell lung cancer cells. We demonstrated that LZ-101 induced mitochondrial-mediated apoptosis by increasing Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c (Cyt c) release and apoptosis-inducing factor (AIF) transposition in A549 cells. Further research illuminated that LZ-101 induced apoptosis was related to the activation of FOXO3a/Bim pathway. Moreover, we found that LZ-101 increased the stability of FOXO3a by blocking autophagy-dependent FOXO3a degradation. In vivo, LZ-101 exerted a remarkable antitumor activity with high safety in xenograft model inoculated A549 tumor through the same mechanism as in our in vitro study. Our findings indicated that LZ-101 induces mitochondrial apoptosis and stabilizes FOXO3a by blocking autophagy flux.

Enhanced energy metabolism plays important roles in the growth and survival of cancer cells. Here, we investigated the mechanisms of a newly synthesized flavonoid, GL-V9, in the inhibition of glycolysis and the induction of apoptosis of human breast cancer cell lines MDA-MB-231 and MCF-7 cells. We find that hexokinase II (HKII) plays important roles in the anticancer effects of GL-V9. GL-V9 not only downregulate the expression of HKII in MDA-MB-231 and MCF-7 cells, but also induce dissociation of HKII from voltage-dependent anion channel (VDAC) in mitochondria, resulting in glycolytic inhibition and mitochondrial-mediated apoptosis. The dissociation of HKII from mitochondria is attributed to GSK-3β-induced phosphorylation of mitochondrial VDAC. Our in vivo experiments also show that GL-V9 significantly inhibits the growth of human breast cancer due to activation of GSK-3β and inactivation of AKT. Thus, GL-V9 induces cytotoxicity via regulation of HKII binding to mitochondria. Our work demonstrates the significance of metabolic regulator in cancer growth and offers fresh insight into the molecular basis for the development of flavonoid as novel agents for the treatment of breast carcinoma.

Identifier

FIDC008851

Recommended Citation

Guo, Yongjian, "Structural Basis for Chloroperoxidase Catalyzed Enantioselective Epoxidations and Mechanisms of Selected Anticancer Drug Induced Apoptosis" (2019). FIU Electronic Theses and Dissertations. 4312.
https://digitalcommons.fiu.edu/etd/4312