Theses and Dissertations - Department of Chemistry & Biochemistry

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Disproving a fifty-five year old myth: Chromium the essential element
(University of Alabama Libraries, 2014) Love-Rutledge, Sharifa Tahirah; Vincent, John B.; University of Alabama Tuscaloosa
Over fifty years ago chromium was proposed to have an essential biochemical role in the metabolism of carbohydrates and lipids, and subsequently its status as an essential element was widely accepted. Unfortunately, these studies confused the pharmacological effects of large, supra-nutritional doses of Cr for nutritional effects. Recent research has firmly established that chromium is not an essential or conditionally essential element for mammals but has effects on insulin sensitivity and cholesterol levels only at pharmacologically relevant doses. However, the mechanism of these effects in rodent models of obesity-associated insulin resistance and type 1 and type 2 diabetes at a molecular level has not been elucidated, although a direct effect on insulin signaling cascade is suggested by current data. This research addresses recent studies that demonstrated that chromium is not essential but pharmacologically active through the use of purified rat diets with varying concentrations of chromium. Urinary chromium excretion in response to an insulin challenge is not a biomarker for Cr status, the effect Cr supplementation on tissue metal concentrations and that Cr is not a conditionally essential element for diabetics as increased Cr urinary excretion in diabetics reflects an increase in absorption using Cr51 tracer studies.
DNA complexes containing novel aromatic residues
(University of Alabama Libraries, 2016) Dong, Wenzhao; Woski, Stephen A.; University of Alabama Tuscaloosa
The investigation of DNA complexes containing novel aromatic residues was performed. In the first part of this work, a series of novel nucleosides possessing a C1’-carboxamide linkage between the aryl moiety and the sugar group were successfully introduced into a single strand DNA oligonucleotide. The results of the thermal denaturation studies indicate that the incorporation of the modified nucleosides into DNA complexes destabilizes the DNA duplexes. However, the “bulged” complexes are only slightly destabilized and they are the most stable complexes among all the DNA complexes containing novel aromatic residues. This suggests that the carboxamide motif may be a general method for the insertion of non-natural residues into DNA for applications such as spectroscopic probes. The second part of this study involves a seven step synthesis of novel aryl C-nucleosides. The aromatic residues are directly linked to the deoxyribose moieties through a carbon-carbon connection instead of the original carbon-nitrogen glycoside bond. Three novel C-nucleosides containing 4-substituted phenyl residues were successfully synthesized by following this synthesis scheme. The isomer problem involved in the multi-step synthesis of aryl C-nucleoside was resolved and as a result, the β-aryl C-2′-deoxynucleoside can be successfully separated from the α-aryl C-2′-deoxynucleoside. The synthesized aryl C-nucleoside can be introduced into a DNA oligonucleotide as a non-natural nucleobase. The third part of this research was focused on the determination of the structure of DNA oligonucleotide duplexes containing aryl C-nucleoside using 2D NMR techniques and computational methods. 2D NMR experiments including COSY and NOESY were performed, followed by resonance assignment and structure calculation to construct the preliminary 3D structure of DNA oligonucleotide duplex containing aryl C-nucleoside. Due to the limitation of the obtained restrains from NMR experiment, the study of molecular modeling has been performed to compensate the ambiguous of the preliminary structure. Conflicts between the calculated duplex structure and the data from the NMR experiment were observed, so an alternate possible structure of hairpin was proposed. The results of thermal denaturation study and molecular modeling may indicate that the hairpin structure is more preferred than the duplex structure for the non-natural DNA oligonucleotide containing aryl C-nucleoside.
Effects of transition metal cationization on peptide dissociation by mass spectrometry
(University of Alabama Libraries, 2011) Watson, Heather Malone; Cassady, Carolyn J.; University of Alabama Tuscaloosa
Peptide sequencing is fundamental to understanding a protein's structure and function. The field of proteomics is dedicated to how these aspects relate to human health and disease. Unfortunately, the majority of peptides and proteins are not fully sequenced. In mass spectrometry, this is often due to spectral complications and incomplete fragmentation. There is a need to develop new sample preparation techniques or dissociation methods to increase sequence information. The dissociation of transition metal-cationized peptides by collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-transfer collisionally activated dissociation (ETcaD) has been investigated in a quadrupole ion trap (QIT). The resulting mass spectra provide a wealth of information about the primary structures of the peptides. Using transition metal ions as cationizing reagents proves beneficial to peptide sequencing by CID and, in some cases, is better than the analysis of protonated species. For instance, spectra obtained from CID of singly and doubly charged Cu(II)-heptaalanine ions, [M + Cu - H]^+ and [M + Cu]^2, are complementary and together provide cleavage at every residue and no neutral losses. This contrasts with protonated heptaalanine, [M + H]^+, which results in fewer backbone cleavages by CID and does not allow sequencing of the first three residues. Multiply charged precursor ions are required in order to carry out ETD and ETcaD. This can be problematic for acidic or neutral peptides. This work demonstrates that addition of transition metals as a cationizing reagent allows peptides to be submitted to ETD and ETcaD that do not otherwise form multiply charged precursors. ETD spectra were less complex than those produced by CID. ETcaD increases backbone cleavages for all samples studied relative to ETD. In addition, complexes that result in very few cleavages by CID are cleaved at every residue when submitted to ETcaD. Evidence for macrocyclic metallated a- and b-ions is found in ETD and ETcaD spectra in the form of nonsequential product ions. The sequence (pEEEEGDD) of the peptide component of biologically derived low-molecular-weight chromium binding substance (LMWCr) is obtained as a result of extensive mass spectrometric studies. LMWCr is proposed to be involved in carbohydrate metabolism. The sequencing of the peptide component of LMWCr by MS represents a potentially significant milestone towards understanding the pharmacological role of chromium at a molecular level.
Functional and regulatory mechanisms in alpha-isopropylmalate synthases
(University of Alabama Libraries, 2014) Casey, Ashley Kay; Frantom, Patrick A.; University of Alabama Tuscaloosa
The allosteric regulation of a protein is where the binding of a molecule at a distal site affects the physical and chemical properties at the binding site. A model system for studying allosteric mechanism is isopropylmalate synthase isolated from Mycobacterium tuberculosis (MtIPMS). MtIPMS catalyzes a Claisen-like condensation between acetyl-CoA and ketoisovalerate to form the products isopropylmalate and CoA, which is the first committed step in the biosynthesis of L-leucine. L-Leucine acts as a slow-onset feedback inhibitor binding 50 Å from the active site in the regulatory domain. Structural studies of MtIPMS indicate that a flexible loop becomes more ordered upon L-leucine binding. Alternate amino acid inhibitors and site-directed mutagenesis results indicate this flexible loop plays a role in the slow-onset mechanism of MtIPMS. Kinetically, L-leucine acts as a V-type inhibitor, lowering V_max for the reaction while K_m values remain relatively unchanged. A decrease in V_max could be caused by a decrease in the rate of a chemical step or product release. Results from rapid-reaction kinetics and kinetic isotope effects indicate that the rate-limiting step shifts from product release to hydrolysis upon the binding of L-leucine. Hydrogen/deuterium exchange experiments indicated that upon L-leucine binding a helix in the active site cavity undergoes a conformational change suggesting that it could be involved in the allosteric mechanism of MtIPMS. The results from site-directed mutagenesis studies indicate that this active site helix is not involved in the allosteric mechanism of MtIPMS. Isopropylmalate synthase isolated from Francisella novicida (FnIPMS) shares a sequences identity of 26% with MtIPMS over 526 residues. This is the first report of a monomeric IPMS to date. The kinetic parameters of FnIPMS are comparable to that of MtIPMS. However, the K_i value is approximately 150-fold higher than that of MtIPMS. Kinetic isotope effects also indicate that hydrolysis is the rate-limiting step in the presence of L-leucine.
Hydrogen deuterium exchange mass spectrometry for protein-protein interaction and structural dynamics
(University of Alabama Libraries, 2013) Singh, Harsimran; Busenlehner, Laura S.; University of Alabama Tuscaloosa
Hydrogen deuterium exchange mass spectrometry has emerged as an important technique to probe protein structure and conformational dynamics. The rate of exchange of hydrogen with deuterium by the peptide backbone is dependent on the solvent accessibility, extent of hydrogen bonding in secondary structural elements and protein dynamics. The extent and the rate of deuterium incorporation are affected by changes in protein structure, interaction with ligand, protein-protein interaction and environmental factors such as pH and temperature. These conformational changes can be global and/or local. The increase in the mass is used to localize the deuterium incorporation after pepsin digestion of the protein and analysis by electrospray ionization mass spectrometry. In this dissertation traditional HDX-MS and a new deuterium trapping assay were used to probe the interaction sites between E. coli cysteine desulfurase SufS and acceptor protein SufE. SufS and SufE form an important part of the SUF pathway, essential for the biosynthesis of Fe-S clusters under oxidative stress and iron depletion conditions. In addition, SufE is known to stimulate SufS cysteine desulfurase activity, but the mechanism is unknown. The HDX-MS results show that the regions affected by the SufS-SufE interaction are dependent on the catalytic intermediate states of the two proteins. HDX-MS was also used to probe the conformational changes resulting upon persulfuration of SufS of Cys364 in the active site. The persulfuration of SufS not only affected regions in the active site cavity, but also had other conformational changes in more distal regions. Based on our findings a model for the interaction SufS and SufE was proposed. A mechanism for the enhancement of SufS cysteine desulfurase activity upon interaction with SufE was also postulated. In all this work demonstrates that hydrogen deuterium exchange mass spectrometry and the deuterium trapping methodology optimized for this system can be easily and effectively used to study the protein-protein interactions and the accompanying changes in structural dynamics for other proteins. Deuterium trapping was demonstrated to be fast, sensitive and reliable method to deduce the changes in solvent accessibility between two or more states of a protein. Both techniques can easily be applied to large number of protein complexes to determine the regions of interaction as well as gain mechanistic information not available through traditional methods such as X-ray crystallography and NMR.
Inhibition of an E. coli DNA glycosylase, MutM, by non-native metals
(University of Alabama Libraries, 2013) An, Mier; Busenlehner, Laura S.; University of Alabama Tuscaloosa
Non-native metals are well recognized carcinogens; however, most exhibit low mutagenicity. One route by which metals could contribute to carcinogenesis is by inhibition of crucial DNA repair processes. The protein targets and mechanism of inhibition, however, are not fully understood. DNA repair proteins that contain zinc finger motifs are potential targets because of their high affinity for metal ions. Insight into the ability of non-native metals to displace the native metal, zinc, and the mechanism they use to inhibit protein function is needed to fully understand this pathway¡¦s contribution to metal-induced cancer. In this dissertation, we probe MutM, an Escherichia coli zinc finger–——containing DNA glycosylase/AP lyase that excises oxidized guanine bases, 8-oxoguanine, from double stranded DNA. We identify that Zn(II)–——, Cd(II)–—— and Co(II)–——MutM complexes coordinate metal ions in the zinc finger motif in a 1:1 stoichiometric ratio. We demonstrate, for the first time, that Cd(II)binding to the MutM zinc finger affects the recognition of 8-oxoguanine containing DNA and inhibits the glycosylase activity, the first step in the mechanism. However, Co(II)–——MutM retains most of the native enzymatic activity, demonstrating the specificity for certain non-native metals. Furthermore, we characterize the conformational and dynamic changes of MutM caused by Cd(II) binding that contribute to the loss of glycosylase activity. This is the first study to relate non-native metal induced changes in structure of zinc finger DNA repair proteins to the mechanism of metal inhibition.
Investigation of protein dynamics in glycosyltransferases with the GT-B structural fold
(University of Alabama Libraries, 2019) Chen, Wen; Frantom, Patrick A.; University of Alabama Tuscaloosa
Carbohydrate transfer reactions are significant for organisms and are involved in a variety of functions in cells. This complicated system is controlled by enzymes including glycosyltransferases (GTs). GTs catalyze sugar transfer reactions. There are mainly two structural folds adopted by GTs, GT-A and GT-B. Structures of the GT-B enzymes are well conserved, especially in the C-terminal domain. GT-Bs are believed to undergo a conformational change upon binding substrates. This indicates that protein dynamics of GT-B members is a key for the study of the whole family. To better understand the commonly shared conformational change by GT-B enzymes, backbone amide hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) is utilized to characterize protein motion in solution. We report results of HDX-MS experiments to determine conformational changes of two representatives of GT-B enzymes, MshA from Corynebacterium glutamicum (CgMshA) and Heptosyltransferase I (HepI). HDX-MS analysis of CgMshA suggests a third conformation of CgMshA in solution with uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) bound. It indicates that the UDP-GlcNAc complex might be in a loose form compared with UDP complex, and the UDP release step might be rate determining for the reaction. Moreover, structural and dynamic studies are combined with bioinformatic results for CgMshA to predict the sequence/structure/dynamic relationships for the GT4 family. In addition, protein dynamics of HepI in the apo form is studied and compared to CgMshA. HDX-MS analysis of HepI suggests that the regions in the outer layer of HepI exhibit flexibility, which predicts a slight domain rotation in HepI. The flexible regions of HepI might participate in the potential conformational change. Other contributions of an additional domain are investigated in this dissertation in terms of protein activity and mechanism. By studying isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS, respectively), the role of the LeuA dimer regulatory domain in substrate selectivity is determined. Overall, we report a more complex role for the LeuA dimer regulatory domain in substrate selectivity through catalytic modulations rather than selectivity through differential binding as a result of extensive co-evolution between the catalytic and regulatory domains.
Investigation of protein-protein interactions in the suf pathway for fe-s cluster assembly in escherichia coli
(University of Alabama Libraries, 2018) Kim, Dokyong; Frantom, Patrick A.; University of Alabama Tuscaloosa
Fe-S cluster cofactor biogenesis is critical to the survival of bacterial pathogens and is carried out by multiprotein biosynthetic pathways. In E. coli, the SUF system is one of three distinct pathways for Fe-S cluster biosynthesis and is utilized under stress conditions. SufS is a group II cysteine desulfurase in the SUF system and requires its partner protein, SufE to mobilize persulfide from L-cysteine. To better understand the molecular details of how SufS and SufE interact, we applied backbone amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with biochemical and biophysical assays. We report results of HDX-MS experiments aimed at identifying changes in the protein dynamics of SufS in complex with the activated variants of SufE and upon persulfuration of SufS. HDX-MS analysis of SufED74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Importantly, ITC studies show that SufEapo binds to SufSapo in a two-site model with negative cooperativity. However, the modified SufED74R binds with a one-site model and a 10-fold increase in affinity. Furthermore, SufED74R exhibits a stoichiometry of 0.57 suggesting that it only binds to one monomer of SufS at a time. These results point toward an additional level of regulation through a half-sites mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by HDX-MS identified two active site peptides and two peptides at the dimer interface of SufS that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions. Three evolutionarily conserved residues at the dimer interface were investigated by alanine scanning mutagenesis. Two variants resulted in 6-fold increases in the value of KSufE, confirming a functional role. The identification of conformational changes at the dimer interface and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to group II cysteine desulfurase systems.
Investigation of regulatory and functional diversity in an enzyme superfamily
(University of Alabama Libraries, 2015) Kumar, Garima; Frantom, Patrick A.; University of Alabama Tuscaloosa
Understanding the evolution of functional and regulatory diversity in enzyme superfamilies addresses a fundamental biochemical problem by improving our ability to identify and exploit structure/function relationships. It opens up the possibility of engineering naturally occurring enzymes and designing new scaffolds for user defined goals. In an attempt to achieve this goal the DRE-TIM metallolyase superfamily has been investigated using bioinformatic and biochemical tools. Analysis of one of the member subgroups, the Claisen condensation-like (CC-like) subgroup, identified the presence of an interesting pattern of functional and regulatory diversity. The CC-like subgroup has ~4300 sequences that catalyze the condensation of acetyl-CoA with six different -keto acids. While some sequentially similar members of this subgroup exhibit distinct substrate specificities, some members with low sequence identities display identical activities. Though the underlying causes of these phenomena are still unknown, evolution of either regulatory and/or functional mechanisms could have generated these discrepancies. To explore diversity in the regulatory mechanisms, two evolutionarily distinct versions of -isopropylmalate synthase (IPMS) enzymes were analyzed. IPMS from Methanococcus jannaschii (MjIPMS) was investigated, and compared with the well characterized IPMS from Mycobacterium tuberculosis (MtIPMS). Isotope effects revealed the conservation of the mechanism of regulation in these different versions of IPMS enzymes. The presence of identical feedback regulation mechanisms in distinct enzymes indicates the complexity of identifying structure/function relationships in multidomain allosteric enzymes. To understand the functional diversity in the CC-like subgroup, IPMS and citramalate synthase (CMS) from Methanococcus jannaschii, MjIPMS and MjCMS, respectively were investigated. MjIPMS and MjCMS share ~50% sequence identity and exhibit distinct substrate specificities for -keto acids. While rational design of substitutions to modulate the active site architecture provided some insight into the mechanism of substrate selectivity for MjIPMS, the mechanism of substrate selection is still unknown for MjCMS. The MjCMS active site was further explored by employing directed evolution tools involving irrational design of substitutions and genetic selection for IPMS activity. Irrational substitutions have been able to deliver initial candidates of MjCMS variants with slightly altered substrate selectivity. Characterization of these libraries should provide significant insight into the mechanism of functional diversity in the DRE-TIM metallolyase superfamily.
Investigations of conserved structure-function relationships and enzymatic evolution
(University of Alabama Libraries, 2021) Conte, Juliana Victoria; Frantom, Patrick A.; University of Alabama Tuscaloosa
The E.coli SUF pathway is the iron-sulfur cluster biogenesis pathway responsible for assembling iron-sulfur clusters during times of oxidative stress and iron starvation. SufS is the cysteine desulfurase enzyme responsible for the acquisition of sulfur and subsequent transfer to transpersulfurase SufE. Recent studies have provided several crystal structures of SufS including two variants displaying stalled intermediates of the desulfurase reaction. Combining the structural data with biochemical investigations has uncovered several key elements in SufS activity. SufS is a homodimer proposed to use a half-sites mechanism involving conformational changes and communication through dimer interface interactions. Despite the significant contributions made to understanding SufS function, questions still remain about the specific roles of several structural elements involved in activity and regulation. Investigations discussed herein are aimed at probing structural components of SufS, including the beta hairpin structure. The beta hairpin makes up one wall of the active site of the opposite monomer. The proposed regulatory mechanism of SufS uses the beta hairpin dynamics to control access to the active site. In chapter 2 beta hairpin variants are shown to significantly disrupt SufS structure. Variants were unable to bind the PLP cofactor essential to catalysis and instability of the variants resulted in protein aggregation as demonstrated with size exclusion chromatography analysis. Chapter 3 focuses on single substitutions of residues located in and around the active site including Asn99, Arg56, and Arg359. Kinetic assays were conducted to determine defects in desulfurase activity by measuring the rate of alanine production, the product released by the desulfurase reaction. Asn99 variants exhibited a ten-fold decrease in turnover number, confirming Asn99 does play a role in generating an optimal environment for activity. This is likely due to the hydrogen bonds Asn99 forms with the glycine residues in the loop at the base of the beta hairpin, potentially contributing to the regulation of dynamics. Arg56 is located in a dynamic loop above the active site and was previously proposed to contribute to catalysis by hydrogen bonding with the sulfhydryl of the substrate cysteine. Kinetic analysis of both R56A and R56K variants is consistent with Arg56 having a significant role in SufS activity. It is possible that this residue is responsible for the deprotonation of SufE C51 and facilitating persulfide transfer. Kinetic analysis of the Arg359 variants suggest a role in cysteine binding and positioning the substrate for bond cleavage. The work presented here contributes to the current understanding of the SufS mechanism and may be more broadly applicable to other cysteine desulfurases.
Iron coordination and protein-protein interactions of the protein frataxin
(University of Alabama Libraries, 2014) Gentry-Dye, Leslie; Busenlehner, Laura S.; University of Alabama Tuscaloosa
Frataxin is a mitochondrial iron metallochaperone that transports ferrous iron to proteins that require it for function. This dissertation research explores the iron binding properties of human frataxin and how frataxin interacts with the mitochondrial [Fe-S] cluster scaffold Isu2 to assemble [Fe-S] clusters. Friedreich's ataxia (FA) is a neurodegenerative progressive limb and gait ataxia that is caused by an exaggerated GAA triplet codon repeat that results in depleted levels of the iron metallochaperone frataxin. Depleted levels of frataxin have a two-fold consequence. The first is that the mitochondria do not have a way to bind and transport iron to proteins that require iron for function. The second is that the cell interprets this as an iron shortage and imports more iron into the mitochondria. As a result, there is both iron overload (caused by having excess non-bioavailable iron in ferric aggregates in the mitochondria) and iron deficiency (since this iron cannot be mobilized for [Fe-S] cluster assembly). Frataxin coordinates ferrous iron and transports it to Isu2 for the assembly of [Fe-S] clusters. In this dissertation, human frataxin Fe2+ coordination was characterized and applied to further study how frataxin interacts with Isu2 for iron transfer and [Fe-S] cluster assembly. This research supports that mature human frataxin coordinates 3 ferrous iron ions and interacts with Isu2 in the same vicinity of Fe2+ coordination for the stimulation of [Fe-S] cluster assembly and provides insight into the cause of FA.
Mass spectrometry studies of peptides cationized by trivalent metal ions
(University of Alabama Libraries, 2018) Commodore-Botoklo, Juliette Joan; Cassady, Carolyn J.; University of Alabama Tuscaloosa
The field of proteomics is dedicated to understanding how a protein’s structure and function relates to human health and disease. Peptide sequencing by mass spectrometry is important to the proteomics movement. Unfortunately, sequencing of many peptides and proteins, such as those with residues containing acidic and neutral side chains, can be difficult. Acidic side chains undergo facile deprotonation that make analysis challenging and can hinder formation of positive mode ions. New methods of sample preparation and dissociation techniques are needed to increase sequence information. This dissertation includes an extensive study of the effects on electron transfer dissociation (ETD) mass spectrometry of biological and model acidic non-phosphorylated and phosphorylated peptides adducted to trivalent lanthanide metal cations. Mass spectra contained herein provide abundant information about the primary structure of peptides. The ETD process requires multiply positively charged ions that can be difficult to obtain with acidic peptides. This work demonstrates that addition of trivalent lanthanide metal cations allows highly acidic peptides to be analyzed by ETD by forming multiply positively charged precursor ions by electrospray ionization (ESI). Using trivalent lanthanide cations as ionizing agents yields extensive sequence information for highly acidic peptides including definitive identification of phosphorylation sites. Peptides forming [M + Met + H]4+ and [M + Met]3+ generate full sequence coverage in many cases, but [M + Pr – H]2+ generates less sequence coverage. (Met is the trivalent metal cation.) The spectra contain primarily a mix of non-metallated and metal adducted c- and z- ions. All metallated product ions incorporate at least two acidic sites or a highly acidic phosphoresidue, which strongly suggests that the trivalent metal cation coordinates with residues that contain highly acidic side chains. All trivalent lanthanide cations are suitable for sequencing highly acidic peptides except europium and radioactive promethium. ETD spectra contain high signal-to-noise ratios making identification of product ions straightforward. Sequence coverage generally improves with increasing peptide chain length. Trivalent chromium enhances protonation of neutral peptides, which is important to ETD analysis. ESI conditions, particularly drying and nebulizing gas pressures are critical to formation of [M + 2H]2+ by neutral peptides.
Potential health benefits of chromium supplementation
(University of Alabama Libraries, 2019) White, Pandora Eula; Vincent, John B.; University of Alabama Tuscaloosa
Trivalent chromium, (Cr(III)), has been used for over 50 years as a “micronutrient”. However, chromium has been shown not to be an essential element. The four studies conducted for this dissertation research attempt to better illuminate how Cr(III) functions in the body. Chapter 2 explores chromium effects on colorectal cancer. In order to explore this link, the effects of Cr(III) compounds were investigated in male and female FVB/NJ mice with azoxymethane-induced colorectal cancer. Cr(III) was found to not have a significant beneficial effect on azoxymethane-induced colorectal cancer. Glucocorticoids, such as dexamethasone, are anti-inflammatory drugs that treat conditions such as arthritis. However, over time the constant use of these drugs impairs wound healing. Cr(III) has been proposed to enhance levels of insulin like growth factor 1 (IGF-1) and increase wound healing. Chapter 3 explores the ability of various Cr(III) compounds to enhance wound healing in C57BL6/JNarl mice receiving dexamethasone. Wound recovery rates, morphological differences and amount of IGF-1 present were determined. Cr(III) was found to not significantly enhance wound healing rates or IGF-1 levels. Bitter melon (BM) has been used in Asia and some parts of Africa as a prophylactic against diabetes. Although research has shown that bitter melon may reduce the effects of diabetes, the exact mechanism, as the case of Cr(III), is unknown. Chapter 4 explores the effects of chromium and bitter melon in insulin-resistant and type 2 diabetic Sprague Dawley rats. The combination of BM and Cr had no beneficial effects on type 2 diabetes or insulin resistance. However, BM tended to reduce glucose levels but negated effects of Cr(III) on insulin resistance in the diabetic rats. Finally, the effects of chromium supplements on farm animals has drawn considerable attention in the last four decades. Thus, a systematic review of the effects of Cr(III) on chickens was undertaken. With the exception of studies on cold-stressed laying hens, the results of studies of Cr supplementation of chickens, whether laying hens or broilers, are too inconsistent for any conclusions to be drawn other than supplementation with Cr led to the acculmulation of Cr in tissues.
The role of outer-sphere residues and substrate-binding at the 3-mercaptopropionic acid dioxygenase (3mdo) active site: a combined spectroscopic and computational investigation
(University of Alabama Libraries, 2021) York, Nicholas James; Pierce, Brad S.; University of Alabama Tuscaloosa
Thiol Dioxygenases (TDOs) are non-heme iron enzymes that catalyze the O2-dependent oxidation of thiol-bearing amino acid derivatives to their corresponding sulfinic acid. Recently, considerable work has been focused on this class of enzymes, as sulfur metabolite imbalances have been correlated with neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and motor neuron disease. The active site of this enzyme class is comprised of a mononuclear Fe(II) bound by three protein derived histidine residues. A conserved feature among structurally characterized TDOs is a sequence of serine, histidine, and tyrosine residues adjacent to the iron active site, deemed the SHY motif. By far, the most well studied TDO is mammalian cysteine dioxygenase (CDO) which oxidizes L-cysteine to cysteine sulfinic acid with high substrate-specificity. Unlike CDO, bacterial 3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av3MDO) is a promiscuous TDO that oxidizes a variety of thiol substrates. Given both enzymes show similar active site geometries, the drastic difference in substrate specificity is not well understood. This dissertation aims to address two unresolved topics among Av3MDO and TDOs in general. The first is the mode in which native substrate, 3MPA, binds to Av3MDO. Arguments have been made for both bidentate coordination through the substrate thiolate and carboxylate and monodentate through the thiolate only. Herein, crystallographic, spectroscopic, and computational studies are used to determine the mode of 3MPA coordination to the iron active site. Bidentate coordination was observed for a crystal structure of bound inhibitor, 3- hydroxypropionic acid, which was used as a basis for computational models of bidentate 3MPA coordination. These models showed agreement with spectroscopic data using either nitric oxide or cyanide as spectroscopic probes. The second aspect investigated is the role of the SHY motif in the active site. For Av3MDO, alterations to the SHY motif have been shown to drastically attenuate activity (kcat), catalytic efficiency (kcat/KM), and formation of NO-bound ES complex. However, the interactions between the SHY motif and iron active site are not fully understood. Spectroscopic studies presented herein reveal the flexible hydrogen bonding capabilities of Tyr159 in the SHY motif and its direct influence on the electronic structure of the iron active site.
The roles of iron and cadmium in human health
(University of Alabama Libraries, 2014) Wang, Yu; Busenlehner, Laura S.; University of Alabama Tuscaloosa
The trace transition metals in humans are divided into two groups, the essential metals and the non-essential/non-native heavy metals. This dissertation research explores the interactions of two transition metals, iron and cadmium, with protein targets to understand their effects on human health. Iron is an important essential metal and is a component of two inorganic cofactors, heme and Fe/S clusters. Disruption of heme and Fe/S cluster cofactor assembly causes downstream protein dysfunction, oxidative stress, and cellular damage. Many diseases, such as the neurodegenerative disease Friedreich's ataxia (FRDA), are caused by the inability to synthesize Fe/S clusters. FRDA is the result of decreased expression of the mitochondrial protein frataxin; however, its exact function is unclear. In this dissertation, a Schizosaccharomyces pombe fission yeast strain was generated in which the yeast frataxin homologue fxn1 was overexpressed to determine what the function(s) of frataxin is through the affected pathways. Based on this study, we demonstrated that S. pombe Fxn1 overexpression elevated the activities of Fe/S enzymes through the up-regulation of Fe/S cluster synthesis, which led to imbalanced iron metabolism, mitochondrial dysfunction and oxidative stress. This research supports that mitochondrial Fxn1 up-regulates the efficiency of Fe/S cluster assembly and provides insight into the cause of FRDA. Besides diseases caused by dysregulation of essential metals, there are diseases related to chronic exposure to heavy metals. The heavy metal cadmium is linked to breast cancers, but with unknown mechanisms. One proposed mechanism is that Cd2+ activates the estrogen receptor &alpha (hERα) transcriptional regulator by binding to the protein and mimicking the conformational effects of the hormone estrogen. We utilized hydrogen/deuterium exchange mass spectrometry to analyze the structural changes of the hERα ligand binding domain upon estradiol or Cd2+ binding. Estradiol binding leads to conformational changes in the dimer interface, the estradiol binding cavity, and the loop between helix H11 and H12. Cadmium demonstrated similar conformational changes at the dimer interface and helix H12. This is the first direct evidence that hERα LBD undergoes structural changes upon Cd2+ binding that are similar to that caused by hormone binding, lending support for this potential mechanism of Cd2+-induced carcinogenesis.

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