Essential Divalent Cation Research Articles

Multilayered alginate–hydrogel-functionalized zeolite based on ionic cross-linking composed with a certain Ca2+ and Mg2+ ratio for ammonium adsorption

To enhance ammonium removal in wastewater treatment, a novel adsorbent, multilayered ionic cross–linked alginate–hydrogel–metal-coated zeolite (Al-H-M-Z), was synthesized. Essential divalent cations, 2.60 mmol of Ca2+ and Mg2+, were incorporated into Al-H-M-Z (10 g) in a Ca2+:Mg2+ ratio of 0.73:1.85, facilitating strong alginate–polymer interactions and significantly enhancing the adsorption efficiency. Langmuir and Freundlich isotherm analyses revealed that Al-H-M-Z had a maximum adsorption capacity of 56.5 ± 8.78 mg g−1 and an adsorption intensity of 0.703 ± 0.122, which were 7.24-fold and 1.59-fold higher than those of natural zeolite (7.80 ± 1.09 mg g−1 and 0.444 ± 0.254), respectively. These enhanced properties are attributed to the modified structural and surface chemistry. The Al-H-M-Z coating exhibited a 4.76-fold increase in surface area (3.91 m2 g−1), an 8.94-fold increase in micropore volume (17.8 × 10−3 cm3 g−1), and a 4.22-fold increase in pore diameter (4.14 nm) when compared to the traditional Al-only coating. For applicability, Al-H-M-Z and Z achieved ammonium removal rates of 7.94% and 5.61%, respectively, in actual fish farm wastewater, representing a 1.41-fold improvement in adsorption efficiency after the coating process.

Effects of zinc in podocytes and cortical collecting duct in vitro and Dahl salt-sensitive rats in vivo

Zinc is one of the essential divalent cations in the human body and a fundamental microelement involved in the regulation of many cellular and subcellular functions. Experimental studies reported that zinc deficiency is associated with renal damage and could increase blood pressure. It was proposed that zinc dietary supplementation plays a renoprotective role. Our study aimed to investigate the effects of zinc on intracellular signaling in renal cells and explore the correlation between dietary zinc and the progression of salt-induced hypertension. The impact of extracellular zinc concentrations on two different kidney epithelial cell types - podocytes and principal cells of the cortical collecting duct (CCD), was tested. In podocytes, a rise in extracellular zinc promotes TRPC6 channel-mediated calcium entry but not altered intracellular zinc levels. However, we observe the opposite effect in CCD cells with no alteration in calcium levels and steady-state elevation in intracellular zinc. Moreover, prolonged extracellular zinc exposure leads to cytotoxic insults in CCD cells but not in podocytes, characterized by increased cell death and disrupted cytoskeletal organization. Next, we tested if dietary zinc plays a role in the development of hypertension in Dahl salt-sensitive rats. Neither zinc-rich nor deficient diets impact the regular development of salt-sensitive hypertension. These results suggest specialized roles for zinc in renal function, implicating its involvement in proliferation and apoptosis in CCD cells and calcium signaling and cytoskeletal dynamics modulation in podocytes. Further research is required to elucidate the detailed mechanisms of zinc action and its implications in renal health and disease.

Biology of calcium homeostasis regulation in intestine and kidney.

Calcium (Ca2+) is an essential divalent cation involved in many bodily functions including bone composition, cell growth and division, blood clotting, and muscle contraction. The bone, intestine, and kidneys are important to the maintenance of Ca2+ homeostasis. Ninety-nine percent of body Ca2+ is stored in the skeleton as hydroxyapatite. The small, and to a lesser extent the large intestine absorbs Ca2+ from the diet. Once in the circulation, Ca2+ is filtered by the glomerulus and the majority, >95%, is reabsorbed along the nephron. The remainder is excreted in the urine. Two general (re)absorptive pathways contribute to the vectorial transport of Ca2+ across renal and intestinal epithelia: 1) a paracellular pathway, which is reliant on claudins in the tight junction of epithelium and the electrochemical gradient and 2) a transcellular pathway, which requires different influx, intracellular buffering/shuttling and basolateral efflux mechanisms, to actively transport Ca2+ across the epithelial cell. Blood Ca2+ levels are maintained by hormones including parathyroid hormone, 1,25-dihydroxyvitamin D3, fibroblast growth factor 23 and through effects of Ca2+-sensing receptor (CaSR) signaling. Disruption of Ca2+ homeostasis can result in altered blood Ca2+ levels and/or hypercalciuria, the latter is a phenomenon closely linked to the formation of kidney stones. Genetic alterations affecting renal Ca2+ handling can cause hypercalciuria, an area of expanding investigation. This review explores the molecular mechanisms governing Ca2+ homeostasis by the intestine and kidneys and discusses clinical aspects of genetic disorders associated with Ca2+-based kidney stone disease.

Characterization of a MHYT domain-coupled transcriptional regulator that responds to carbon monoxide.

The MHYT domain, identified over two decades ago for its potential to detect diatomic gases like CO, O2or NO, has awaited experimental validation as a protein sensory domain. Here, we characterize the MHYT domain-containing transcriptional regulator CoxC, which governs the expression of the cox genes responsible for aerobic CO oxidation in the carboxidotrophic bacterium Afipia carboxidovorans OM5. The C-terminal LytTR-type DNA-binding domain of CoxC binds to an operator region consisting of three direct repeats sequences overlapping the -35 box at the target PcoxB promoter, which is consistent with the role of CoxC as a specific transcriptional repressor of the cox genes. Notably, the N-terminal transmembrane MHYT domain endows CoxC with the ability to sense CO as an effector molecule, as demonstrated by the relief of CoxC-mediated repression and binding to the PcoxB promoter upon CO exposure. Furthermore, copper serves as the essential divalent cation for the interaction of CO with CoxC, thereby confirming previous hypothesis regarding the role of copper in the gas-sensing mechanism of MHYT domains. CoxC represents the prototype of a novel subfamily of single-component LytTR transcriptional regulators, characterized by the fusion of a DNA-binding domain with a membrane-bound MHYT sensor domain.

Impacts and Industrial Applications of Phytic Acid and Phytase

Phytic acid is an antinutritional compound that chelates P and essential divalent cations such as Fe, Ca, and Zn in cereals and major staple crops such as wheat, maize, rice, and sorghum. As a result, these cations cannot be absorbed by monogastric animals or humans: phytic acid has an inhibitory effect on nutrient uptake and its levels are negatively correlated with protein and starch digestibility. However, phytic acid can be degraded by the action of the enzyme phytase. Phytase plays important roles in the degradation of phytic acid and in increasing the nutritional quality of staple foods. Microbial phytase is a versatile enzyme that is beneficial for humans, animals, the environment, and the industry. In this review, we summarise the interaction of phytic acid with micronutrients, various approaches to enhancing the nutritional profile of staple foods by reducing the phytic acid content, and current knowledge of microbial-based phytase as a potential reducer of phytic acid.

Magnesium Status and Ca/Mg Ratios in a Series of Children and Adolescents with Chronic Diseases.

Magnesium (Mg) is an essential divalent cation involved in various enzymatic reactions that regulate vital biological functions. The main goal was to evaluate Mg status and its association with nutritional indicators in 78 children and adolescents with chronic diseases. We assessed anthropometric, biochemical, diet, body composition, and bone densitometry valuations. Serum Mg and Ca levels were determined using the standardized method and diet calcium (Ca) and Mg consumption by a prospective 72 h diet survey. Mean serum Ca (9.9 mg/dL), Mg (2.08 mg/dL) dietary Ca (102% DRI: Dietary Reference Intake), and Mg intake (105% DRI) were normal. A total of 45% had hypomagnesemia, 12% had hypermagnesemia, and 26% and 24% had inadequate and high Mg intake, respectively. Only 6% of patients had poor Mg intake and hypomagnesemia, and 54% and 90% of our series had an elevated serum Ca/Mg ratio > 4.70 (mean 4.79) and a low Ca/Mg intake ratio < 1.70 (mean 1.06), respectively. Both Ca/Mg ratios were linked with the risk of developing other chronic conditions such as cardiovascular disease, type 2 diabetes, syndrome metabolic, and even several cancers. Therefore, 79% of children and adolescents with chronic diseases were at elevated risk of having abnormal Mg status and developing other chronic illnesses.

Structural mechanism of TRPM7 channel regulation by intracellular magnesium

Zn2+, Mg2+ and Ca2+ are essential divalent cations implicated in many metabolic processes and signalling pathways. An emerging new paradigm is that the organismal balance of these cations predominantly depends on a common gatekeeper, the channel-kinase TRPM7. Despite extensive electrophysiological studies and recent cryo-EM analysis, an open question is how the channel activity of TRPM7 is activated. Here, we performed site-directed mutagenesis of mouse TRPM7 in conjunction with patch-clamp assessment of whole-cell and single-channel activity and molecular dynamics (MD) simulations to show that the side chains of conserved N1097 form an inter-subunit Mg2+ regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg2+ binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg2+ favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg2+, representing a new structure–function relationship not yet explored among TRPM channels.

Magnesium in aging and aging-related disease

Magnesium (Mg2+) is an essential divalent cation in human body. Its balance is tightly controlled via a balanced interplay among intestinal absorption, storage, and renal excretion, involving multiple transporters across cell membrane that regulate Mg2+ influx and efflux. Mg2+ is involved in a variety of physiological and pathological processes such as enzymatic reactions, energy metabolism, cell proliferation, apoptosis, oxidative stress, and inflammation. In particular, Mg2+ contributes to the molecular hallmarks of aging. Emerging evidence demonstrates that altered Mg2+ status has been associated with many aging-related diseases, including cancer, cardiovascular disease, neurodegenerative disease, musculoskeletal function, metabolic syndrome, and COVID-19. In this review, we focus on Mg2+ and its association with molecular hallmarks of aging. We also summarize recent findings supporting an important role of Mg2+ in aging-related disease including the COVID-19 pandemic.

Corrosion-resistant Mg(OH)2/Mg-Fe layered double hydroxide (LDH) composite films on magnesium alloy WE43

BackgroundA new synthesis approach was developed for Mg-Fe layered double hydroxide (LDH) film on oxidised magnesium (Mg) alloy WE43 surface by cathodic electrodeposition. MethodsThe synthesised Mg(OH)2 surface layer provides essential divalent cations and a foundation for the direct growth of an Mg-Fe LDH film through the electrodeposition process with trivalent cations containing solution. FindingsThe cross-section analysis results disclosed that the Mg(OH)2 layer altered the typical morphology of LDH film, leading to structured LDH nanoplates with a thickness of approximately 3.5 µm. The formation of structured LDH nanoplates resulted from the perpendicular arrangement of the ab-faces of the crystallites to the WE43 surface, as seen from Field Emission Scanning Electron Microscopy (FE-SEM) images. Electrochemical tests in Hank's Balanced Salt Solution (HBSS) report the Ecorr value increased by 0.24 V and a decrease in icorr by one order of magnitude of Mg(OH)2/Mg-Fe LDH composite films against its substrate. Furthermore, the impedance modulus values of Mg(OH)2/Mg-Fe LDH composite films can achieve a magnitude of three times greater than its substrate. These results indicate that the Mg-Fe LDH film can effectively capture the corrosion anions during corrosion through the anion-exchange process, making it a promising surface modification for Mg alloy.

A protective superhydrophobic Mg–Zn–Al LDH film on Surface-Alloyed Magnesium

A novel approach was developed to fabricate a protective superhydrophobic LDH film on a Zn–Al alloyed surface of AZ31 Mg alloy. The alloyed surface layer not only provided essential divalent and trivalent cations and a base for the direct growth of a Mg–Zn–Al LDH film in a sodium nitrate hydrothermal solution, but also offered additional corrosion and mechanical protection for the substrate magnesium alloy. Further modification with low surface energy lauric acid (LA) could change the micro/nano hierarchical topographic surface of the LDH film from hydrophilicity to super-hydrophobicity. The long-term anti-corrosion performance, super-hydrophobicity, wear resistance, chemical stability and adhesion strength of the LA-modified LDH film on the surface-alloyed AZ31 were experimentally verified.

Differential responses of growth, photosynthesis, oxidative stress, metals accumulation and NRAMP genes in contrasting Ricinus communis genotypes under arsenic stress.

Effect of arsenate [As(V)] on biomass, photosynthetic rate, stomatal conductance, transpiration, oxidative stress, accumulation of As, Fe, Zn, Cu and Mn and expression of NRAMP genes was investigated in As(V) tolerant and sensitive genotypes of bioenergy crop Ricinus communis. As(V) treatments (100 and 200 μM) led to significant reduction in root and leaf biomass, photosynthetic rate, stomatal conductance and transpiration in GCH 2 and GCH 4 genotypes but no significant change or increase was observed in WM and DCH 177 genotypes. No significant difference was observed in hydrogen peroxide content and lipid peroxidation in As(V)-treated tolerant genotypes compared to control, whereas these parameters enhanced significantly in As(V)-treated sensitive genotypes. GCH 2 accumulated around two times As in leaves and showed significant reduction in concentration of Zn and Mn in the leaves and roots due to 200 μM As(V) treatment compared to WM. NRAMP genes are critical for uptake and distribution of essential divalent metal cations, photosynthesis and controlled production of reactive oxygen species in plants. RcNRAMP2, RcNRAMP3 and RcNRAMP5 genes showed differential expression in response to 200 μM As(V) in GCH 2 and WM suggesting that NRAMP genes are associated with differential responses of WM and GCH 2 genotypes to As(V) stress.

Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.

Characterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana, an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of AtIspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of AtIspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of AtIspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.

Autosomal-Recessive Intellectual Disability with Cerebellar Atrophy Syndrome Caused by Mutation of the Manganese and Zinc Transporter Gene SLC39A8

Manganese (Mn) and zinc (Zn) are essential divalent cations used by cells as protein cofactors; various human studies and animal models have demonstrated the importance of Mn and Zn for development. Here we describe an autosomal-recessive disorder in six individuals from the Hutterite community and in an unrelated Egyptian sibpair; the disorder is characterized by intellectual disability, developmental delay, hypotonia, strabismus, cerebellar atrophy, and variable short stature. Exome sequencing in one affected Hutterite individual and the Egyptian family identified the same homozygous variant, c.112G>C (p.Gly38Arg), affecting a conserved residue of SLC39A8. The affected Hutterite and Egyptian individuals did not share an extended common haplotype, suggesting that the mutation arose independently. SLC39A8 is a member of the solute carrier gene family known to import Mn, Zn, and other divalent cations across the plasma membrane. Evaluation of these two metal ions in the affected individuals revealed variably low levels of Mn and Zn in blood and elevated levels in urine, indicating renal wasting. Our findings identify a human Mn and Zn transporter deficiency syndrome linked to SLC39A8, providing insight into the roles of Mn and Zn homeostasis in human health and development.

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

The multiple nucleotide–divalent cation binding modes ofSaccharomyces cerevisiaeCK2α indicate a possible co-substrate hydrolysis product (ADP/GDP) release pathway

CK2 is a ubiquitous and conserved protein kinase in eukaryotic organisms and is important in many biological processes. It is unique in maintaining constitutive activity and in using both ATP and GTP as phosphor donors. In this study, crystal structures of recombinant Saccharomyces cerevisiae CK2α (scCK2α) complexed with GMPPNP, ATP and AMPPN with either Mg2+ or Mn2+ as the coordinated divalent cation are presented. The overall structure of scCK2α shows high similarity to its homologous proteins by consisting of two domains with the co-substrate lying in the cleft between them. However, three characteristic features distinguish scCK2α from its homologues. Firstly, the Lys45-Glu53 and Arg48-Glu53 interactions in scCK2α lead Lys50 to adopt a unique conformation that is able to stabilize the γ-phosphate of the co-substrate, which makes the existence of the `essential divalent cation' not so essential. The multiple nucleotide-divalent cation binding modes of the active site of scCK2α are apparently different from the two-divalent-cation-occupied active site of Zea mays CK2α and human CK2α. Secondly, conformational change of Glu53 in scCK2α-AMPPN breaks its interaction with Lys45 and Arg48; as a result, the co-substrate binding pocket becomes more open. This may suggest a clue to a possible ADP/GDP-release pathway, because the NE1 atom of the Trp in the `DWG motif' of CK2α forms a hydrogen bond to the O atom of Leu212, which seems to make ADP release by means of the `DFG-in flip to DFG-out' model found in most eukaryotic protein kinases impossible. Coincidentally, two sulfate ions which may mimic two phosphate groups were captured by Arg161 and Lys197 around the pocket. Mutagenesis and biochemical experiments on R161A and K197A mutants support the above proposal. Finally, scCK2α is unique in containing an insertion region whose function had not been identified in previous research. It is found that the insertion region contributes to maintaining the constitutively active conformation of the scCK2α catalytic site, but does not participate in interaction with the regulatory subunits.

Prolactin‐stimulated ubiquitination of the zinc transporter ZnT2 regulates zinc secretion

Zinc (Zn), an essential divalent cation, is required for over 300 biological processes including DNA and protein synthesis and intracellular signaling. The Zn transporter ZnT2 regulates Zn export from mammary epithelial cells (MECs) into milk during lactation. Tight regulation of ZnT2 is critical to ensure adequate Zn transfer to the infant yet maintain Zn homeostasis in MECs. Prolactin (PRL) regulates nutrient production and secretion from MECs. PRL signaling is deactivated through ubiquitination and degradation of its receptor (PRLr). Herein, we report that after an initial increase, PRL decreased Zn secretion in endogenous and ZnT2‐overexpressing MECs. This resulted from ZnT2 internalization and ubiquitination, which targeted ZnT2 for proteasome‐dependent degradation. Mutagenesis identified two N‐terminal lysine residues (K4 and K6) necessary for ubiquitin attachment. Mutation of these lysine residues to arginine (K4R and K6R) inhibited ubiquitination and degradation, which confined ZnT2 to the cell membrane and maintained Zn secretion. These findings indicate that PRL‐mediated activation of ubiquitin‐dependent ZnT2 degradation represses Zn secretion from MECs. This suggests that PRL‐stimulated ubiquitination regulates ZnT2 function to maintain optimal cell function in mammary cells and provides insight into novel mechanisms through which ubiquitination regulates ion transport.

Exploration and Mining of the Bacterial Terpenome

Tens of thousands of terpenoids are present in both terrestrial and marine plants, as well as fungi. In the last 5-10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin, the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself, have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of "genome mining". In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg(2+), that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate. The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes. Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria), as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been elucidated. In addition, studies of terpenoid biosynthetic gene clusters have revealed a wealth of previously unknown oxidative enzymes, including cytochromes P450, non-heme iron-dependent dioxygenases, and flavin monooxygenases. We have shown that these enzymes catalyze a variety of unusual biochemical reactions, including two-step ketonization of methylene groups, desaturation-epoxidation of secondary methyl groups, and pathway-specific Baeyer-Villiger oxidations of cyclic ketones.

Structure of a Nudix hydrolase (MutT) in the Mg(2+)-bound state from Bartonella henselae, the bacterium responsible for cat scratch fever.

Cat scratch fever (also known as cat scratch disease and bartonellosis) is an infectious disease caused by the proteobacterium Bartonella henselae following a cat scratch. Although the infection usually resolves spontaneously without treatment in healthy adults, bartonellosis may lead to severe complications in young children and immunocompromised patients, and there is new evidence suggesting that B. henselae may be associated with a broader range of clinical symptoms then previously believed. The genome of B. henselae contains genes for two putative Nudix hydrolases, BH02020 and BH01640 (KEGG). Nudix proteins play an important role in regulating the intracellular concentration of nucleotide cofactors and signaling molecules. The amino-acid sequence of BH02020 is similar to that of the prototypical member of the Nudix superfamily, Escherichia coli MutT, a protein that is best known for its ability to neutralize the promutagenic compound 7,8-dihydro-8-oxoguanosine triphosphate. Here, the crystal structure of BH02020 (Bh-MutT) in the Mg(2+)-bound state was determined at 2.1 Å resolution (PDB entry 3hhj). As observed in all Nudix hydrolase structures, the α-helix of the highly conserved `Nudix box' in Bh-MutT is one of two helices that sandwich a four-stranded mixed β-sheet with the central two β-strands parallel to each other. The catalytically essential divalent cation observed in the Bh-MutT structure, Mg(2+), is coordinated to the side chains of Glu57 and Glu61. The structure is not especially robust; a temperature melt obtained using circular dichroism spectroscopy shows that Bh-MutT irreversibly unfolds and precipitates out of solution upon heating, with a T(m) of 333 K.

Structural Insights into the Catalytic Mechanism of Bacterial Guanosine-diphospho-d-mannose Pyrophosphorylase and Its Regulation by Divalent Ions

GMP catalyzes the formation of GDP-Man, a fundamental precursor for protein glycosylation and bacterial cell wall and capsular polysaccharide biosynthesis. Crystal structures of GMP from the thermophilic bacterium Thermotoga maritima in the apo form, in complex with the substrates mannose-1-phosphate or GTP and bound with the end product GDP-Man in the presence of the essential divalent cation Mg(2+), were solved in the 2.1-2.8 A resolution range. The T. maritima GMP molecule is organized in two separate domains: a N-terminal Rossman fold-like domain and a C-terminal left-handed beta-helix domain. Two molecules associate into a dimer through a tail-to-tail arrangement of the C-terminal domains. Comparative analysis of the structures along with characterization of enzymatic parameters reveals the bases of substrate specificity of this class of sugar nucleotidyltransferases. In particular, substrate and product binding are associated with significant changes in the conformation of loop regions lining the active center and in the relative orientation of the two domains. Involvement of both the N- and C-terminal domains, coupled to the catalytic role of a bivalent metal ion, highlights the catalytic features of bacterial GMPs compared with other members of the pyrophosphorylase superfamily.

ATP, calcium and magnesium levels in platelets of patients with primary fibromyalgia

Objectives To evaluate the intracellular levels of the high energy adenosine triphosphate nucleotide ATP and essential divalent cations, calcium and magnesium, in platelets of patients affected by primary fibromyalgia syndrome (FMs). Design and method Platelet ATP and cation concentrations were measured in 25 patients affected by FMs and 25 healthy volunteers through a chemiluminescent and a fluorimetric assay, respectively. Results Significant lower ATP levels were observed inside platelets of FM patients (fmol ATP/plt: 0.0169 ± 0.0012 vs. healthy controls, fmol ATP/plt: 0.0306 ± 0.0023, mean ± SEM) (⁎⁎⁎ P < 0.0001). A trend towards higher calcium concentrations ( P = 0.06) together with significant increased magnesium levels were also reported in platelets of patients by comparison with controls ( P = 0.02). Conclusions This preliminary study suggests that disturbances in the homeostasis of platelet ATP metabolism-signaling and calcium-magnesium flows might have a relevance in the pathogenesis of FMs.