Author ORCID Identifier
0000-0002-5596-4806
Date of Award
8-31-2026
Document Type
Campus Access Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Integrative Biosciences
First Advisor
Daniel P. Dowling
Abstract
Cofactor-dependent biocatalysts catalyze a wide range of chemical transformations with high selectivity under mild conditions. Their dependence on tightly regulated cofactors allows them to achieve both high efficiency and specificity, making them especially powerful in biological systems. Beyond their native roles in metabolism, these enzymes occupy a unique position at the interface of enzymology, structural biology, and applied chemistry. As a result, they have become valuable tools for designing therapeutics, removing environmental pollutants, and building complex molecular libraries for drug discovery. This dissertation investigates the structural and functional characteristics of three cofactor-dependent biocatalysts: a metal-dependent human inorganic pyrophosphatase 1 (hsPPA1) as a cancer drug target, a two-component flavin-dependent monooxygenase SfnG as a biocatalyst for sulfur bioremediation, and a radical S-adenosyl-L-methionine enzyme NeoN as a potential tool for synthesizing novel carbohydrate-based libraries. Cancer is one the leading causes of death worldwide, highlighting the need for developing novel targeted therapies. Enzymes are central in the complex biology of cancer and are important targets for anti-cancer drug design. Human inorganic pyrophosphatase 1 (hsPPA1) is a ubiquitous metalloenzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). hsPPA1 maintains cellular phosphorus homeostasis by removing the PPi released during the synthesis of biomolecules, thus making biosynthetic reactions thermodynamically favorable. Therefore, hsPPA1 is critical to cellular metabolism, and its upregulation and overexpression have been implicated in cancer cell proliferation and metastasis. Thus, hsPPA1 is being explored as a promising enzyme target for the development of novel anti-cancer drugs. To support structure-based inhibitor design, in chapter 3 of this dissertation, we determined the X-ray crystal structures of hsPPA1 in multiple liganded states: with magnesium or lithium ions bound and a fluoride-inhibited structure complexed with magnesium and pyrophosphate ions. These structures show that hsPPA1 adopts the conserved oligonucleotide/oligosaccharide (OB) fold, and substrate binding induces subtle conformational changes in the active site residues. Structure of hsPPA1 with lithium ions bound, combined with functional studies, showed that the structure represents a Li(+)-inhibited state of the enzyme. In addition, the functional assays on the variants of the residue D118 and phosphate ions revealed details on the chemical mechanism of hsPPA1 and the dual effects of phosphate on hsPPA1 activity. Finally, we report on the effect of magnesium concentrations and reducing agent on hsPPA1 activity. Together, these findings provide structural and functional insight into hsPPA1, as well as establish a framework for structure-based inhibitor design. Anthropogenic sulfur emissions have disrupted the global sulfur cycle, posing a significant threat to the ecosystem and human health. Under inorganic sulfur limiting conditions, certain bacteria, including Pseudomonads have evolved alternative pathways that allow the assimilation of organosulfur pollutant compounds as sulfur sources. Central to this process are two-component flavin dependent monooxygenases (TC-FMOs), which catalyze key transformations in the breakdown of compounds, such as dimethyl sulfone (DMSO2) under sulfur starvation conditions. In Pseudomonads, DMSO2 is converted to sulfite via the action of the TC-FMOs SfnG, MsuC and MsuD, and the FMN reductase MsuE, encoded by the msu and sfn operons. In chapter 4 of this dissertation, we investigated the structural and biochemical basis of the class C TC-FMO SfnG in Pseudomonas fluorescens, which catalyzes conversion of DMSO2 to methanesulfinate (MSI-). The preliminary crystal structure of SfnG with an alternative substrate revealed the critical conformational changes that occur in the active site lid region upon the binding of FMN and a longer alkylsulfone, ethylmethyl sulfone (EMSO2). The structure shows the binding of EMSO2 may accompany movement of active site residues to accommodate longer substrates. In addition, binding studies with DMSO2 and longer alkylsulfones revealed the ordered binding and substrate promiscuity of SfnG. Lastly, binding studies on the active site mutants, Y60A and Q53E, highlight the critical importance of these residues in FMN and substrate binding. Together, these studies present a deeper understanding of the dimethyl sulfone monooxygenase SfnG to accommodate and catalyze its native as well as alternative substrates and provides the opportunities for exploring these enzymes for potential sulfur bioremediation purposes. Carbohydrates present significant synthetic challenges due to their structural complexity and dense functionalization, often requiring protecting groups that leads to increased synthesis steps and reduced efficiency. Enzymatic approaches that allow selective isomerization and epimerization are potential alternatives for producing rare and unnatural sugars, although many remain limited by thermodynamic constraints and poor selectivity. Radical SAM enzyme epimerases offer a promising solution by catalyzing highly selective, radical-dependent transformations that can overcome these limitations. However, the underlying mechanism of rSAM epimerases remains poorly understood due to limited structural information. In chapter 5 of this dissertation, we focus on the characterization and structural analysis through computational methods of an rSAM epimerase NeoN, involved in the final step of neomycin antibiotic biosynthesis. Biochemical, bioinformatic, and computational structural analyses classify NeoN as a member of the Twitch subfamily, containing an auxiliary iron-sulfur cluster vital for catalysis. These findings expand our understanding of radical-medicated enzyme catalysis and provide a foundation for the experimental structure elucidation of NeoN. It also provides opportunities for potentially exploring rSAM enzymes for the efficient synthesis of rare sugars for drug development. Together, the structural and functional characterization of the three cofactor-dependent biocatalysts highlights their diverse catalytic strategies and broad application potential. The metal-dependent hsPPA1 provides insights into its catalytic mechanism and lays the foundation for structure-based anticancer drug development. The TC-FMO SfnG advances our knowledge of bacterial sulfur metabolism and offers opportunities for bioremediation of organosulfur pollutants. The rSAM enzyme NeoN provides the basis for its structural characterization with potential applications in expanding the carbohydrate-based libraries. Collectively, this dissertation presents the importance of studying the structural and biochemical aspects of enzymes to broaden their applications in drug design, environmental remediation, and synthetic biology.
Recommended Citation
Mawani, Jayata Shailesh, "Deciphering structural and biophysical characteristics of cofactor dependent biocatalysts: Mg(2+)-dependent human inorganic pyrophosphatase 1, flavin-dependent dimethylsulfone monooxygenase, and radical S-adenosylmethionine-dependent neomycin C epimerase" (2026). Graduate Doctoral Dissertations. 1184.
https://scholarworks.umb.edu/doctoral_dissertations/1184
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