Graduation Semester and Year

2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Kayunta Johnson-Winters

Abstract

The focus of this research is to elucidate the catalytic mechanism of F420-dependent enzymes using F420-dependent glucose-6-phosphate dehydrogenase (FGD) from Mycobacteria tuberculosis (Mtb) and F420H2:NADP+ Oxidoreductase (Fno) from Archaeoglobus fulgidis (A. fulgidis) as models. To investigate the mechanism of these enzymes, our group primarily utilizes UV-vis spectrophotometric techniques, in order to conduct steady-state and pre steady-state kinetic experiments. We also use fluorescence binding, and kinetic isotope effects methods in order to provide an in-depth understanding of these enzymes. This conglomeration of work will help to serve as model systems to enzymes who exhibit similar behavior, especially the understudied F420-dependent enzymes. Chapter 1 outlines the structural and spectral properties of the F420 cofactor, along with the phylogenetic distribution of the cofactor amongst species. Additionally, chapter 1 summarizes the literature and previously conducted work on wild-type Fno (wtFno). The second half of chapter 1 serves as a preface for the investigation of amino acids at the subunit interface that give rise to inner-subunit communication within Fno. This work is discussed in detail in Chapter 4. Chapter 2 is a comprehensive review focused on FGD. This chapter highlights the key findings of FGD in the past decade, while comparing FGDs from various sources. Historically, FGD from Mycobacteria smegmatis and Mtb have been studied extensively due to the major health relevance of the pathogenic bacteria, Mtb, which is the causative agent of tuberculosis disease (TB). FGD is an excellent therapeutic target for multiple drug resistant and extreme drug resistant forms of Mtb. In recent years, FGD has been identified and crystallized in Rhodococcus jostii. A detailed comparison of the Rhodococcus jostii RHA1 FGD structure to the Mtb structure is discussed in this chapter, along with newly identified FGDs that display promiscuity towards a variety of sugar-6-phosphates, known as FSDs. The amino acid sequences, along with preliminary steady-state kinetic analysis with a variety of sugar-6-phosphates of the FSDs from Nocardia and Cryptosporangium arvum are discussed. Until our work on F420-dependent enzymes, rigorous enzymological investigation of these enzymes were non-existent. Chapter 2 highlights the kinetic characterization of wild-type FGD (wtFGD) and FGD variants from Mtb and the updated FGD catalytic mechanism. Our previous work has led to many unanswered questions concerning the FGD mechanism. Therefore, chapter 3 investigates the role of active site residues, E13, H40 and H260. We have utilized the techniques mentioned above and have extended our analysis to the global analysis using KinTek Global Kinetic Explorer. The purpose of the global analysis of the data is to identify any potential intermediates, while getting a handle on the intrinsic rate constants before the first turnover. The pre steady-state kinetic data revealed an F420-based intermediate. Additionally, H40 is assisted by E13, suggesting these residues act as a catalytic dyad in a similar fashion as the NADP-dependent G6PDs. Chapter 4 aims to explore the functionality of the three conserved amino acids that were previously suggested to aid in G6P binding. We have characterized, K198, K259, and R283 by generating a library of variants, which include, K198Q, K259A, R283A, R283K, and a double mutant K259A/R283A using site-directed mutagenesis. We then kinetically characterized these variants using the spectroscopic techniques discussed above. Chapter 5 focuses on the inner-subunit communication of a separate F420-dependent enzyme, known as Fno. Fno regulates NADPH production within archaea. This enzyme catalyzes the reversible reduction of NADP+ to NADPH via a direct hydride transfer reaction from the reduced F420 cofactor to the C-4 position of NADP+, producing NADPH. Our 2016 kinetic investigation of wtFno reveals that the enzyme exhibits negative cooperativity and half-site reactivity. From this work, we then identified several amino acids at the subunit interface that could potentially play a role in how the two subunits communicate and therefore regulate NADPH production. These residues include: R186, T192, T09, S190, and H133. Kinetic studies on these mutated residues at the subunit interface reflected changes in cooperativity, from negative to no cooperativity. Additionally, several variants lost half-site reactivity. Fno is the first F420-dependent enzyme to display a change in cooperativity provides great insight into NADPH regulation.

Keywords

F420-dependent enzymes

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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