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Author ORCID Identifier

https://orcid.org/0000-0001-6181-5113

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2020

Month Degree Awarded

September

First Advisor

Prof. Michael J Maroney

Second Advisor

Prof. Michael J Knapp

Third Advisor

Prof. Min Chen

Fourth Advisor

Prof. Scott C. Garman

Subject Categories

Biochemistry | Inorganic Chemistry

Abstract

Cellular nickel pools, comprised of static and labile pools of nickel complexes, play important roles in maintaining nickel homeostasis in various organisms (microbes, fungi, and plants), which utilize it as a cofactor of one or more nickel enzymes that catalyze specific reactions and are essential for their proper growth and survival in various ecological niches. Like other metals, tight regulation of cellular nickel levels is critical to prevent toxic effects of nickel deprivation, nickel overload, and ‘free’ nickel. While more static nickel pools include nickel tightly bound to nickel-dependent enzymes, nickel in the labile pool is exchangeable and weakly bound to either nickel chaperones or low-molecular-weight (LMW) ligands, as is the case for many other transition metals. The role of nickel chaperones in enzyme maturation and activation is being extensively investigated, but the importance of cellular LMW complexes in the process remains largely unknown.

In this work, I investigated the role of labile nickel pools (both non-proteinaceous and proteinaceous ligands) in the maturation of nickel-dependent enzymes - Nickel Superoxide Dismutase (NiSOD) from Streptomyces coelicolor, and urease and Ni, Fe-hydrogenase from Helicobacter pylori. For the maturation of NiSOD, no chaperone has been characterized for nickel delivery to the active site. Using biochemical assays, mass spectrometry, and spectroscopic methods, I provide compelling evidence that biologically relevant, non-proteinaceous, LMW ligand complexes of nickel with L-histidine are inevitable for the proper maturation of the enzyme. In Helicobacter pylori, which is a human pathogen, there are two nickel dependent enzymes – urease and Ni, Fe hydrogenase both of which require a proteinaceous labile nickel ligand (common nickel-chaperone), HypA for maturation. HypA interacts with HypB for the maturation of Ni, Fe-hydrogenase, and with UreE for the maturation of urease, but what factors determine its differential interaction with its downstream partner proteins are unknown. I interrogated the flexibility and dynamics of HypA by glycine mutations to perturb the maturation of the two nickel enzymes in the pathogen and using in vitro assays I show that the glycine mutations differentially affect the maturation of urease and Ni, Fe – hydrogenase in the pathogen.

DOI

https://doi.org/10.7275/18847822

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