Expression, isolation, and structural characterization of E. coli DNA repair enzyme MutM

No abstract available. (Published By University of Alabama Libraries

Exposing the Alkanesulfonate Monooxygenase Protein–Protein Interaction Sites

The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein–protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen–deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein–protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein–protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation

His86 from the N‑Terminus of Frataxin Coordinates Iron and Is Required for Fe–S Cluster Synthesis

Human frataxin has a vital role in the biosynthesis of iron–sulfur (Fe–S) clusters in mitochondria, and its deficiency causes the neurodegenerative disease Friedreich’s ataxia. Proposed functions for frataxin in the Fe–S pathway include iron donation to the Fe–S cluster machinery and regulation of cysteine desulfurase activity to control the rate of Fe–S production, although further molecular detail is required to distinguish these two possibilities. It is well established that frataxin can coordinate iron using glutamate and aspartate side chains on the protein surface; however, in this work we identify a new iron coordinating residue in the N-terminus of human frataxin using complementary spectroscopic and structural approaches. Further, we demonstrate that His86 in this N-terminal region is required for high affinity iron coordination and iron assembly of Fe–S clusters by ISCU as part of the Fe–S cluster biosynthetic complex. If a binding site that includes His86 is important for Fe–S cluster synthesis as part of its chaperone function, this raises the possibility that either iron binding at the acidic surface of frataxin may be spurious or that it is required for protein–protein interactions with the Fe–S biosynthetic quaternary complex. Our data suggest that iron coordination to frataxin may be significant to the Fe–S cluster biosynthesis pathway in mitochondria

The roles of iron and cadmium in human health

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. (Published By University of Alabama Libraries

Functional and regulatory mechanisms in alpha-isopropylmalate synthases

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. (Published By University of Alabama Libraries

Characterization of the roles of phosholipase B1 and the cyclic-AMP (cAMP)-protein kinase A (PKA) pathway in nutritional and osmotic stress response in Schizosaccharomyces pombe

Physiological stress is a reality faced by all cells. Even though these stresses are often disruptive of normal cellular function, cells must adapt to the stressor or perish. This study utilizes the model organism Schizosaccharomyces pombe, commonly known as fission yeast, to study a novel stress response pathway for adaptation to nutrient deprivation and hyperosmotic stress involving phospholipase B1 (Plb1) and components of the cyclic-AMP (cAMP)-protein kinase A (PKA) pathway. A previous study showed that deletion of plb1 confers sensitivity to hyperosmotic stress in the form of potassium chloride (KCl). This phenotype could be rescued by over-expression of components of the cAMP-PKA pathway, and likewise, deletion of these components also lead to KCl sensitivity phenotypes. This study shows that deletion of these genes also correlates with increased fragmentation of mitochondria under conditions of hyperosmotic stress. Interestingly, addition of rotenone or loss of mitochondrial PE synthesis - conditions that have previously been associated with increased mitochondrial fragmentation -exacerbated phenotypes of the plb1mutant. Mitochondrial fragmentation was also accompanied by increased mitophagy in KCl-treated plb1 cells. Cell cycle arrest in G2/M and cytokinesis was observed in KCl-treated cells in which the gene encoding the PKA catalytic subunit pka1 had been deleted. These phenotypes - mitochondrial fragmentation and failure to complete cytokinesis - may be due to deregulation of PS and PE synthesis and cellular distribution. In addition to participating in hyperosmotic stress response, Plb1 and the cAMP-PKA pathway cooperate to respond to nutrient deprivation. The function of the cAMP-PKA pathway in glucose sensing is well established in yeast. Previous studies have suggested a role for phospholipases B (PLBs) in nutrient scavenging, though a limited number of studies have examined how nutrient content affects secretion of these PLBs. In this study, we have found that the secretion of Plb1 is increased in nutrient-poor media. In particular, glucose content greatly affects Plb1 secretion, since incubation in low glucose media highly increases secretion whereas addition of glucose reduces secretion. Since the cAMP-PKA pathway is responsible for detecting glucose in the media, we predicted that deletion of pka1 would lead to increased Plb1 secretion. However, Plb1 secretion was not appreciably altered in a pka1 mutant. Interestingly, pka1 mutant cells did have increased levels of Plb1 protein, and the localization of Plb1 in these cells resembled that seen in glucose-starved cells, suggesting that Plb1 and the cAMP-PKA pathway may interact with regards to glucose sensing. (Published By University of Alabama Libraries

Synthesis and characterization of hierarchically porous metal, metal oxide, and carbon monoliths with highly ordered nanostructure

Hierarchically porous materials are of great interest in such applications as catalysis, separations, fuel cells, and advanced batteries. One such way of producing these materials is through the process of nanocasting, in which a sacrificial template is replicated and then removed to form a monolithic replica. This replica consists of mesopores, which can be ordered or disordered, and bicontinuous macropores, which allow flow throughout the length of the monolith. Hierarchically porous metal oxide and carbon monoliths with an ordered mesopores system are synthesized for the first time via nanocasting. These replicas were used as supports for the deposition of silver particles and the catalytic efficiency was evaluated. The ordered silica template used in producing these monoliths was also used for an in-situ TEM study involving metal nanocasting, and an observation of the destruction of the silica template during nanocasting made. Two new methods of removing the silica template were developed and applied to the synthesis of copper, nickel oxide, and zinc oxide monoliths. Finally, hollow fiber membrane monoliths were examined via x-ray tomography in an attempt to establish the presence of this structure throughout the monolith. (Published By University of Alabama Libraries