Citrate Binding Research Articles

Structure-Guided Discovery of a Potent Inhibitor of the Ferric Citrate Binding Protein FecB in Vibrio Bacteria.

Infections caused by Gram-negative bacteria present a significant risk to human health worldwide. Novel strategies are needed to deal with the challenge caused by drug-resistant bacteria. Here, we report a new approach to combat infections by targeting iron-binding proteins to suppress bacterial growth. We investigated the function of the conserved periplasmic binding protein FecB from Vibrio alginolyticus. FecB was known to play a crucial role in the bacterial growth and to relate with biofilm formation. We then solved the crystal structures and elucidated the binding mechanism of FecB with ferric ion chelated by citrate. The results indicated that FecB binds weakly to one citrate molecule and strongly to the Fe3+-(citrate)2 complex. Based on these results, a structure-based virtual screening approach was conducted against FecB to identify small molecules that block ferric citrate uptake. Further evaluations in vivo and in vitro demonstrated that salvianolic acid C significantly suppressed bacterial growth, indicating that targeting bacterial nutrient absorption is a promising strategy for identifying potential antibacterial drugs.

Structure‐Guided Discovery of a Potent Inhibitor of the Ferric Citrate Binding Protein FecB in Vibrio Bacteria

AbstractInfections caused by Gram‐negative bacteria present a significant risk to human health worldwide. Novel strategies are needed to deal with the challenge caused by drug‐resistant bacteria. Here, we report a new approach to combat infections by targeting iron‐binding proteins to suppress bacterial growth. We investigated the function of the conserved periplasmic binding protein FecB from Vibrio alginolyticus. FecB was known to play a crucial role in the bacterial growth and to relate with biofilm formation. We then solved the crystal structures and elucidated the binding mechanism of FecB with ferric ion chelated by citrate. The results indicated that FecB binds weakly to one citrate molecule and strongly to the Fe3+‐(citrate)2 complex. Based on these results, a structure‐based virtual screening approach was conducted against FecB to identify small molecules that block ferric citrate uptake. Further evaluations in vivo and in vitro demonstrated that salvianolic acid C significantly suppressed bacterial growth, indicating that targeting bacterial nutrient absorption is a promising strategy for identifying potential antibacterial drugs.

Calcium bioaccessibility as affected by strontium. Temperature effect on citrate binding to strontium and calcium alone and in combination

Strontium as a bone health promoter was found to increase the bioaccessibility of essential calcium from solid calcium citrate tetrahydrate in aqueous suspensions. This is unexpected since calcium citrate binds calcium stronger than strontium with ΔH0ass,c = 5.07 kJ⋅mol−1, ΔS0ass,c = 78.0 J⋅mol−1⋅K−1 for 1:1 Ca-Citrate complex formation and ΔH0ass,c = 1.67 kJ⋅mol−1, ΔS0ass,c = 58.3 J⋅mol−1⋅K−1 for 1:1 Sr-Citrate complex formation as determined both electrochemically and by isothermal titration calorimetry. Calcium citrate tetrahydrate has reverse solubility around physiological temperature in contrast to strontium citrate pentahydrate. This difference results in opposite effect of strontium upon calcium ion liberation from calcium citrate suspensions at both low and high temperature compared to physiological temperature, for which strontium was found to stabilize solid calcium citrate tetrahydrate. This effect, confirmed by quantum mechanical calculation and by comparison of spontaneous aqueous supersaturation of calcium citrate and strontium citrate, is suggested to be involved in the positive effect of strontium on bone and teeth health. The conclusions provide a theoretical foundation for the research of molecular mechanisms among calcium, strontium, and citrate ions in relation to bone and teeth health.

Diminishing the Pathogenesis of the Food-Borne Pathogen Serratia marcescens by Low Doses of Sodium Citrate.

Protecting food from bacterial contamination is crucial for ensuring its safety and avoiding foodborne illness. Serratia marcescens is one of the food bacterial contaminants that can form biofilms and pigments that spoil the food product and could cause infections and illness to the consumer. Food preservation is essential to diminish such bacterial contaminants or at least reduce their pathogenesis; however, it should not affect food odor, taste, and consistency and must be safe. Sodium citrate is a well-known safe food additive and the current study aims to evaluate its anti-virulence and anti-biofilm activity at low concentrations against S. marcescens. The anti-virulence and antibiofilm activities of sodium citrate were evaluated phenotypically and genotypically. The results showed the significant effect of sodium citrate on decreasing the biofilm formation and other virulence factors, such as motility and the production of prodigiosin, protease, and hemolysins. This could be owed to its downregulating effect on the virulence-encoding genes. An in vivo investigation was conducted on mice and the histopathological examination of isolated tissues from the liver and kidney of mice confirmed the anti-virulence activity of sodium citrate. In addition, an in silico docking study was conducted to evaluate the sodium citrate binding ability to S. marcescens quorum sensing (QS) receptors that regulates its virulence. Sodium citrate showed a marked virtual ability to compete on QS proteins, which could explain sodium citrate's anti-virulence effect. In conclusion, sodium citrate is a safe food additive and can be used at low concentrations to prevent contamination and biofilm formation by S. marcescens and other bacteria.

Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196

The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity.

Impact of pectin and whey minerals solubilized by lime juice on calcium bioaccessibility in yogurt based snacks

Pectin is used in dairy products to improve texture, but it may also affect calcium absorption. The influence of pectins with different degrees of esterification on calcium bioaccessibility in yogurt snacks enriched with or without whey mineral concentrates and lime juice was quantified using a three-step in vitro digestion including electrochemical determination of free and complex bound calcium. Calcium from whey minerals became bioaccessible under intestinal conditions when combined with lime juice, as a consequence of a large fraction of complex bound calcium by citrate. Pectin improved the texture of the snacks but lowered the calcium bioaccessibility by 6%–16% depending on the degree of esterification through endothermic binding of calcium to carboxylate groups of pectins as evidenced by higher yield stress of the snack bases. Decreasing degree of esterification and increasing blockiness promoted calcium binding, as indicated by the estimated binding sites and association constants. Increasing temperature from 25 °C to 37 °C and pH from 4 to 7 (intestinal conditions) also increased the interactions between calcium and pectins. For low methoxyl pectin, the complex binding of calcium to pectin carboxylates was found more evident than binding to citrate from the lime juice reducing calcium bioaccessibility. For high methoxyl pectin with fewer binding sites, citrate binding kept calcium available for uptake through intestinal digestion. A balanced formulation with whey minerals, lime juice and calcium-insensitive high methoxyl pectin yielded crispy yogurt snacks with high calcium bioaccessibility, which provides a new perspective to design dairy products with high nutritional value. • Pectin improves hardness and crispness of freeze-dried yogurt snacks. • Whey minerals with lime juice increase Ca bioaccessibility by citrate binding. • LM and to a lesser degree of HM pectin decreases Ca bioaccessibility. • LM pectin reduces calcium binding to citrate decreasing Ca bioaccessibility. • HM pectin and whey minerals lead to crispy snacks and high Ca bioaccessibility.

The Multiple Roles of the Active Site Loop of Watermelon Glyoxysomal Malate Dehydrogenase

All malate dehydrogenases have a flexible loop that starts to close over the active site region of malate dehydrogenase when cofactor binds and locks down completing the active site when the dicarboxylic acid substrate binds. As such the active site loop appears to play a role in both substrate specificity and correct alignment of the substrate to allow hydride transfer and proton abstraction/donation depending on the direction of the reaction. Combined with available crystal structures, detailed analysis of the 1smk.pdb structure shows the loop in a series of microstates from fully open to a closed state induced by the inhibitor citrate binding (figure 1), sequence conservation suggests that the loop region contains a 19 amino acid stretch from P119 to N137 using the watermelon glyoxysomal enzyme as reference. Clustal Omega Analysis of malate dehydrogenases from prokaryotes to mammals shows 5 families of conservation of the loop region (figure 2) allowing a series of hypotheses about structure function relationships of specific residues within the sequence to be made which have been tested by a combination of computational and site directed mutagenesis wetlab approaches. In addition to the two canonical arginines involved in substrate binding, we have explored every other residue in the loop region. Overall, mutations in the first half of the loop had small impact on Specific Activity while mutations in the second half of the loop had larger impacts. R130 mutants to E or S had the most impact while mutation to A or Q had lesser effects. Three other residues in particular had striking effects on Oxaloacetate saturation, G121A, L133S and N137. L133 and N137, at the C terminal base of the loop, were hypothesized to play a role in the repulsive interactions between substrate and cofactor in the active site. Consistent with this hypothesis, creation of an L133S or D137A mutant showed that concomitant with a reduction in specific activity to 2% and 5% wildtype (respectively), Km for Oxaloacetate was dramatically increased while Km for NADH was decreased somewhat. Finally, a series of mutations of M128 (Q,I and A) were explored with varying effects on specific activity (Q>I>A) and significant effects on Km for Oxaloacetate but lesser or no effects on Km for NADH. The impact of M128 mutations in particular were further explored computationally using DeepDDG analysis and effects on stability correlated with impact on kinetic properties. Overall these studies provide a detailed picture of the complex interactions the flexible loop makes that contribute to both substrate binding and catalysis.

Chitosan Matrix Encapsulation of α‐Lipoic Acid (LA) Anchored Gold Nanoparticles: A Combined Experimental and Theoretical Study

AbstractIn this article the competitive binding of citrate (2‐hydroxypropane‐1,2,3‐tricarboxylate), chitosan and α‐lipoic acid ((R)‐5‐(1,2‐dithiolan‐3‐yl) pentanoic acid, (LA) are tested on the surface of Gold Nanoparticle (AuNP). The chitosan matrix encapsulated gold nanoparticle resulted in colour reversibility in presence of α‐lipoic acid through partial ligand exchange mechanism. Different experimental analysis including TEM, EDX, spectrophotometry and FTIR were spectrometry performed to check the interaction mechanism and explain the colour reversibility. Binding and colour reversibility mechanism was also supported through the Density functional Theory calculations.

Citrate Binding Buffer

Citrate Binding Buffer

Manipulating the crystallization kinetics and morphology of gypsum, CaSO4·2H2O via addition of citrate at high levels of supersaturation and the effect of high salinity

Citrate ion possesses the right structure to be strongly bound on specific crystal faces of gypsum, CaSO4·2H2O, which results in an increase in the induction time of precipitation, and changes the structure and morphology of the solid. The inhibition of the precipitation kinetics of gypsum, in the stoichiometric reaction of Na2SO4 and CaCl2 was investigated in aqueous solutions with high supersaturation (0.1 M initial reactant concentrations). The induction time was found to be the longest when the inhibitor was present in fully deprotonated form in the solution (pH > 5). The inhibition effect levels off with the increasing inhibitor concentrations (ca. 3 mM). As a result of citrate binding, on the basis of SEM measurements, the morphology of the crystals change from rod-like to plate-like. Addition of high concentration of NaCl (mimicking seawater conditions) was found to facilitate the inhibition, but from the morphological studies, the inhibition mechanism of citrate appears to change under hypersaline conditions (relative to systems without added electrolyte.) From UV and IR spectroscopic measurements, it was found that the inhibitor ended up in the mother liquor after the completion of the precipitation reaction.

Functional analysis of a species-specific inhibitor selective for human Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY).

The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.

Mechanism of Action of Veverimer: A Novel, Orally Administered, Nonabsorbed, Counterion-Free, Hydrochloric Acid Binder under Development for the Treatment of Metabolic Acidosis in Chronic Kidney Disease.

Current management of metabolic acidosis in patients with chronic kidney disease (CKD) relies on dietary intervention to reduce daily endogenous acid production or neutralization of retained acid with oral alkali (sodium bicarbonate, sodium citrate). Veverimer is being developed as a novel oral treatment for metabolic acidosis through removal of intestinal acid, resulting in an increase in serum bicarbonate. Veverimer is a free-amine polymer that combines high capacity and selectivity to bind and remove hydrochloric acid (HCl) from the gastrointestinal (GI) tract. In vitro studies demonstrated that veverimer had a binding capacity of 10.7 ± 0.4 mmol HCl per gram of polymer with significant binding capacity (>5 mmol/g) across the range of pH values found in the human GI tract (1.5-7). Upon protonation, veverimer bound chloride with high specificity but showed little or no binding of phosphate, citrate, or taurocholate (<1.5 mmol/g), which are all anions commonly found in the human GI tract. Administration of veverimer to rats with adenine-induced CKD and metabolic acidosis resulted in a significant increase in fecal chloride excretion and a dose-dependent increase in serum bicarbonate to within the normal range compared with untreated controls. Absorption, distribution, metabolism, and excretion studies in rats and dogs dosed with 14C-labeled veverimer showed that the polymer was not absorbed from the GI tract and was quantitatively eliminated in the feces. Acid removal by veverimer, an orally administered, nonabsorbed polymer, may provide a potential new treatment for metabolic acidosis in patients with CKD. SIGNIFICANCE STATEMENT: Metabolic acidosis is a complication of chronic kidney disease (CKD) as well as a cause of CKD progression. Veverimer is a high-capacity, selective, nonabsorbed, hydrochloric acid-binding polymer being developed as a treatment for metabolic acidosis. Veverimer binds and removes hydrochloric acid from the gastrointestinal tract, resulting in increased serum bicarbonate and the correction of metabolic acidosis. Veverimer is not an ion-exchange resin and does not deliver sodium or other counterions, and so it may be appropriate for patients with CKD with and without sodium-sensitive comorbidities.

ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia

A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.

Novel Tat-Dependent Protein Secretion.

The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the cytoplasmic membrane to the FecI extracytoplasmic function (ECF) sigma factor. In this issue of Journal of Bacteriology, I. J. Passmore, J. M. Dow, F. Coll, J. Cuccui, et al. (J Bacteriol 202:e00541-19, 2020, https://doi.org/10.1128/JB.00541-19) report that the FecR sequence contains both the twin-arginine signal motif and the secretory (Sec) avoidance motif typical of proteins secreted by the twin-arginine translocation (TAT) system. The same study shows that FecR is indeed secreted by Tat and represents a new class of bitopic Tat-dependent membrane proteins.

Design, Synthesis, Computational, and Preclinical Evaluation of natTi/45Ti-Labeled Urea-Based Glutamate PSMA Ligand.

Despite promising anti-cancer properties in vitro, all titanium-based pharmaceuticals have failed in vivo. Likewise, no target-specific positron emission tomography (PET) tracer based on the radionuclide 45Ti has been developed, notwithstanding its excellent PET imaging properties. In this contribution, we present liquid–liquid extraction (LLE) in flow-based recovery and the purification of 45Ti, computer-aided design, and the synthesis of a salan-natTi/45Ti-chelidamic acid (CA)-prostate-specific membrane antigen (PSMA) ligand containing the Glu-urea-Lys pharmacophore. The compound showed compromised serum stability, however, no visible PET signal from the PC3+ tumor was seen, while the ex vivo biodistribution measured the tumor accumulation at 1.1% ID/g. The in vivo instability was rationalized in terms of competitive citrate binding followed by Fe(III) transchelation. The strategy to improve the in vivo stability by implementing a unimolecular ligand design is presented.

ACLY is the Novel Signaling Target of PIP &lt;sub&gt;2&lt;/sub&gt;/PIP &lt;sub&gt;3&lt;/sub&gt; and Lyn in Cancer

ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, the ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells.

Structure of an α-glucuronidase in complex with Co2+ and citrate provides insights into the mechanism and substrate recognition in the family 4 glycosyl hydrolases

Glycosyl hydrolases belonging to the family 4 (GH4) use a unique redox-based NAD+-dependent reaction mechanism involving anionic intermediates and requires a divalent metal ion and reducing conditions for catalytic activity. These enzymes display wide specificity and selectivity for their substrates. However, the structural basis of substrate binding, recognition and specificity remains poorly studied. Here, we report the crystal structure of Thermotoga maritima TmAgu4B, a GH4 α-glucuronidase, in complex with Co2+ and citrate. Analysis of GH4 structures show that the metal ion is present in a conserved octahedral coordination with conserved side chain atoms, the ligand atoms and an invariant water molecule. The data provides the first structural evidence for a metal-activated hydroxide ion that acts as the general base to deprotonate the C3-hydroxyl group of the glycone, a rate-limiting step in the mechanism. Furthermore, the citrate binding mode in the active site is analogous to a bound glucuronide substrate and provides insights into the mode of substrate interaction with the metal ion, the active site residues and, the structural basis of substrate recognition in a GH4 α-glucuronidase.

An allosteric mechanism for potent inhibition of human ATP-citrate lyase.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1-5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have alsodetermined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure-activity relationships of these compounds. This allosteric site greatly enhances the 'druggability' of ACLY and represents an attractive target for the development of new ACLY inhibitors.

Alterations within fumarase C reveal newly observed SS Loop conformations

Fumarase belongs to a larger superfamily of enzymes including aspartase, adenylosuccinate lyase, arginosuccinate lyase, and 3-carboxy-cis, cis-muconate lactonizing enzyme. Each enzyme member shares a common quaternary structure and harbors a flexible active site loop, termed the SS Loop. The SS Loop lies within the fumarase superfamily signature sequence 317GS318SxMxK324xN326xxPxE331 and plays a fundamental role during catalysis. Recently, human fumarase deficiencies have been linked to renal cell carcinoma and benign uterine tumor formation. In this study we site-selectively altered SS Loop residues serine 318, lysine 324, asparagine 326 and glutamic acid 331 to alanine. The fumarase C SS Loop variants were characterized functionally and structurally. Functionally, steady-state kinetic experiments were conducted in the classic Krebs Cycle sense (fumarate to S-malate) and in the reductive biosynthetic direction (S-malate to fumarate). Structurally, the single-site SS Loop variants were analyzed via circular dichroism and x-ray crystallography. The steady-state kinetic results report minimal effect on the S-malate and fumarate Km values, while reducing kcat significantly. We have placed the kcat reduction into two categories: (1) diminished charge-relay influence and (2) SS Loop immobility. Structurally, the SS Loop single-site alanine variants have similar thermal and chemical denaturation profiles. Thus, we suggest the overall homotetrameric quaternary structure, a requisite for a functional multi-subunit active site, is intact. X-ray crystallographic results for both the serine 318 to alanine (S318A) and lysine 324 to alanine (K324A) variants report a newly discovered fumarase C active site architecture. The SS Loop in both the S318A and K324A active sites undertakes two disparate conformations leading to two unique active sites. These two active sites are non-crystallographically related and undertake ordered-open and ordered-closed conformations. Thus, alteration of serine 318 and lysine 324 has facilitated crystallographic order within the SS Loop region. Ordering and closure of the S318A SS Loop has created an electrostatic gate between the multi-subunit active site and an auxiliary single-subunit citrate binding site. Adaptation within the K324A crystal structure includes additionally ordered active site water molecules. Effectively, the newly observed water molecules re-establish electrostatic interactions between serine 98, threonine 100, asparagine 141, histidine 188 and the previously observed active site water. Our structural and functional results further establish the importance of SS Loop conformational mobility within the fumarase superfamily. S318A SS Loop and domain 3 movement lead to closure of the fumarase C active site. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study on the effect of some surface phenomena on the properties of citrate capped cobalt doped ferrites

Magnetic nanoparticles for applications in life sciences require their uniform dispersion in water which involves surface modification with organic hydrophylic molecules. This study was focused on cobalt doped ferrite nanoparticles known mainly for their usefulness in experimental oncologic thermotherapy. The influence of cobalt doping level was analyzed with focus on crystallinity, granularity and saturation magnetization, based mainly on experimental data provided by X-ray diffractometry, Transmission Electron Microscopy, Vibrating Sample Magnetometry, as well as on the mathematical modeling of data resulted from Small Angle X-ray Scattering measurements.The features of spinel crystallization system of citrate capped cobalt ferrite powders were described with X-ray diffractometry while magnetic core morphology and size polydispersity were evidenced by Transmission Electron Microscopy. Small Angle X-ray Scattering, carried out on fluid samples, revealed theoretically different particle associations for iron oxide and cobalt doped nanoparticles that could be discussed assuming different citrate binding in case of irregular surface. Magnetic properties were studied with Vibrating Sample Magnetometry, revealing superparamagnetism in all samples and also coercivity for cobalt doped ferrites; discussion was developed on the influence of surface crystal defects on saturation magnetization. Citrate ion interaction with magnetic core surface was confirmed by FTIR recordings. The interpretation of cobalt influence on the microstructural and magnetic properties of the nanoparticle series of CoxFe3-xO4 (x = 0; 0.25; 0.50; 0.75; 1) was based on different binding of citrate at the surface metal ions for magnetite and cobalt ferrites.