Design, engineering & characterization of a PQQ containing artificial metalloenzyme

Thompson, Peter J

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https://hdl.handle.net/2142/129829 Copy

Description

Title

Design, engineering & characterization of a PQQ containing artificial metalloenzyme

Author(s)

Thompson, Peter J

Issue Date

2025-07-01

Director of Research (if dissertation) or Advisor (if thesis)

Olshansky, Lisa

Doctoral Committee Chair(s)

Olshansky, Lisa

Committee Member(s)

• Gruebele, Martin
• Aksimentiev, Aleksei
• Manesis, Anastasia

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Quant Biology

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Artificial Metalloenzyme
• PqqT
• ArM
• PQQ
• enzyme
• protein engineering
• crystallography

Abstract

The complexity of the reactions catalyzed by enzymes, coupled with the incorporation of metal centers within them, has led chemists to study and exploit the unique environments afforded by metalloproteins. Naturally occurring metalloproteins, however, are often challenging to study due to numerous technical barriers inherent to protein purification and reconstitution protocols. These difficulties are exemplified within the PQQ-dependent alcohol dehydrogenase (ADH) enzymatic machinery of methylotrophic bacteria. For the ADH reaction to proceed within their native bacteria, a three-component system is leveraged where the ADH enzyme requires a partner protein for activation and a subsequent cytochrome redox partner to return the ADH enzyme back to its oxidized state for further reaction. A recent surge of interest into this ADH enzymatic pathway has developed since the 2011 discovery of lanthanide (Ln3+) elements present in the active sites of these PQQ-dependent ADH enzymes. This newfound interest has resurfaced a debate concerning the mechanistic pathway of these ADH enzymes. Analogous Ca2+-dependent ADH enzymes have been known since the 1970s however, due to difficult heterologous expression, multi-component reaction pathway, and challenging product analysis, this mechanistic debate has yet to be fully addressed. Challenges such as these related to the ADH enzymes and others motivate bioinorganic chemists to either create simplified or modified biological scaffolds that possess native or engineered metallocofactor binding sites to study more complex processes. In 2019, a periplasmic binding protein was discovered to co-express with the La3+ dependent ADH proteins. This protein, now known as PqqT, is a proposed PQQ transport protein for the import of the necessary cofactor for the ADH process. While these bacteria have biosynthetic machinery for the creation of PQQ, it has been reported that this pathway is downregulated in the presence of La3+ further supporting PqqT as a putative PQQ binding protein. Our lab adopted this newly discovered protein with special interest in conformational dynamics of proton coupled electron transfer (PCET) within quinone binding proteins. In chapter two, we show the first crystal structure of PQQ bound to WT-PqqT which revealed a binding pocket that shared similarities with the naturally occurring ADH enzymes. These structural similarities served as inspiration for a rational design approach to create a binding pocket that resembles that of the Ca2+- and La3+-dependent ADH enzymes. Our structure-driven mutagenetic study showed that upon introducing K115D to the PqqT binding pocket not only established preferential binding for La3+ over Ca2+ but also introduced ADH reactivity not present prior to this mutation. With a newly created artificial metalloenzyme (ArM) created within the PqqT scaffold, this protein presented us an opportunity to explore the mechanistic behavior exhibited by this ArM during catalysis. The third chapter explores the mechanistic pathways of the PQQ(La3+) ⸦ K115D-PqqT variant when reacted with para-substituted benzyl alcohol derivatives. The results of the linear free energy relationship (LFER) and kinetic isotope effect (KIE) experiments suggest a hydride transfer contributing to a 30-year debate. Chapter three also explores the structural implications of increasing the binding affinity of PQQ within the K115D-PqqT variant by introducing the mutation Y161W. Despite an increase in affinity, this mutation abolishes the activity of this variant under identical reaction conditions of PQQ(La3+) ⸦ K115D-PqqT. Finally, this chapter shows how the product formation of the ADH reaction performed by PQQ(La3+) ⸦ K115D-PqqT decreases with the introduction of heavier Ln3+ elements. Chapter four starts where chapter three ends by further exploring the implications behind a change in metal identity and it’s influence on product formation. Through electron paramagnetic resonance (EPR) spectroscopy, UV-vis absorption spectroscopy, LFER, and KIE experiments, the work presented here shows that a decrease in product formation may be due to a change in the rate limiting step of the oxidative ADH reaction. Through the tuning of the PQQ cofactor, the Ln3+ elements prove to behave in a capacity beyond acting as a Lewis acid. This thesis concludes by introducing the metal binding capabilities that the PqqT scaffold can accommodate upon further mutational studies. The content presented here shows that K115H variants of PqqT have a specificity toward binding Cu2+ over other transition metals however, through modified metalation strategies, other metals can be incorporated. This work establishes a new ArM construct primed for further catalytic exploration through unique quinone-metal coupling and protein modification.

Graduation Semester

2025-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/129829

Copyright and License Information

Copyright 2025 Peter Thompson

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