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Full text release has been delayed at the author's request until December 15, 2029

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Exploring the Catalysis and HDL-Association Mechanisms of Paraoxonase-1 and Optimization of Protein Sequence Using Consensus Analysis Approach

Chen, Shuhan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1724080084955282

Abstract Details

2024, Doctor of Philosophy, Ohio State University, Chemistry.
Paraoxonase-1 (PON1) is an enzyme universally found in mammalian serum. It has been shown to hydrolyze a very broad range of substrates. The native substrates of this enzyme are likely to be lactones, while it also hydrolyzes aryl esters and organophosphates with lower efficiencies. PON1 also binds to high-density lipoproteins (HDL) in serum, which stabilizes the protein significantly and enhances its activities in vitro. It’s also related to this enzyme’s physiological function in mammalian serum. After being produced in liver cells, PON1 is transferred to HDL in serum and exerts anti-oxidative and anti-inflammatory functions. Loss of serum paraoxonase activities increases the risk of atherosclerosis development. Currently, due to the unstable nature of this protein, the structure information of native human PON1 is still not available. Several recombinant PON1 variants were created with higher stability and solubility. One variant along with several of its mutants (G2E6) was successfully crystalized and its structure was solved. Even though all current research is using G3C9 variant due to its better performance, a lot of information still can be exploited from G2E6 structures considering the high level of homology in their sequences. And thus, numerous mechanistic studies are performed based on this limited structural information. Based on all the available information, it is reasonable to believe that PON1 may adopt different mechanisms for different substrates. For lactonase and aryl esterase activities, key residues include the H115/H134 dyad, which have been hypothesized to serve as proton shuttlers and activate a water molecule for nucleophilic attack. This hypothesis is supported by a lot of mutagenesis and kinetics studies. But for paraoxonase activity, D269 is more likely to be the Lewis base that activates a water molecule for subsequent nucleophilic attack. Out of all the proposed mechanisms, the catalytic calcium ion is always seen as a Lewis acid that coordinates with and activates the substrate. We postulate that the role of the cation may be more than this. By replacing the catalytic calcium ion with different divalent cations that share similar ionic radius, the protein failed to exhibit similar activities as with Ca2+ originally. This suggests that the Ca2+ relies on not just its size and charge but also its chemical properties to facilitate the hydrolysis. The versatile enzymatic activities arise mainly from the similarities in the binding complex structures and plasticity of the enzyme’s active site structure. This can be simply attributed to coincidence, or it indicates that this enzyme is in a middle stage of evolution. This is also the theoretical basis of many directed-evolution studies carried out so far. By introducing certain amount of mutation, several variants were selected with high activities against specific substrate structure or stereochemistry. Studying enzymes with latent abilities, like PON1, can really help us learn more about the protein’s evolvability and potentially about how to engineer an enzyme and optimize the desired functions. Aside from activity promiscuity, another important feature of PON1 is HDL association. Mapping PON1-HDL interaction sites can be challenging. Unlike other protein-protein interactions, it also involves protein-lipid interactions, which adds complexity to research. Currently, it is believed that the binding is mediated by Apolipoprotein AI (ApoAI) and insertion of PON1’s first helix. But our knowledge of the specific binding sites is not comprehensive. Also, simulation studies revealed that HDL binding restrains the plasticity of the active site through forming a H-bond network connecting the active site and binding interface. However, considering the large number of hydrophobic and aromatic residues at the binding region, we hypothesize that the binding mechanism may involve more than just these polar interactions. We mutated 8 surface residues and measured their binding affinities with HDL particles and collected their kinetics data against phenyl acetate and paraoxon. It turned out that their binding affinities with HDL don’t always correlate with its stimulation effect on activities. One single mutation can change either one (binding affinity or stimulation effect) or both aspects. So, to further decipher the binding mechanisms, more advanced approaches will be required. Lastly, one of the biggest obstacles in PON1 research is the difficulty in expressing native human PON1 variants. A large amount of effort has been paid in creating systems for human PON1 expression and purification, but the yield was little. The recombinant PON1 variants exhibit good properties but bear relatively low similarity to human PON1. We attempted to use consensus analysis approach to design a more human-like PON1 variant. This approach helps identify all crucial sites conserved throughout the evolution. It doesn’t necessarily guarantee a positive result, but it definitely provides insights into the sequence and can be a good start for future engineering.
Thomas Magliery (Advisor)
125 p.
Biochemistry
Paraoxonase-1, enzymes, metal-placement, Calcium, HDL, allosteric propagation, consensus analysis, Pf3

Recommended Citations

Citations

  • Chen, S. (2024). Exploring the Catalysis and HDL-Association Mechanisms of Paraoxonase-1 and Optimization of Protein Sequence Using Consensus Analysis Approach [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1724080084955282

    APA Style (7th edition)

  • Chen, Shuhan. Exploring the Catalysis and HDL-Association Mechanisms of Paraoxonase-1 and Optimization of Protein Sequence Using Consensus Analysis Approach. 2024. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1724080084955282.

    MLA Style (8th edition)

  • Chen, Shuhan. "Exploring the Catalysis and HDL-Association Mechanisms of Paraoxonase-1 and Optimization of Protein Sequence Using Consensus Analysis Approach." Doctoral dissertation, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1724080084955282

    Chicago Manual of Style (17th edition)

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© 2024, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.
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