Manganese-containing Enzyme Research Articles

Manganese (III/IV) μ-Oxo Dimers and Manganese (III) Monomers with Tetraaza Macrocyclic Ligands and Historically Relevant Open-Chain Ligands.

The oxygen-evolving complex (OEC) located in photosystem II (PSII) of green plants is one of the best-known examples of a manganese-containing enzyme in nature, but it is also used in a range of other biological processes. OEC models incorporate two multi-dentate nitrogen-containing ligands coordinated to a bis-μ-oxo Mn(III,IV) core. Open-chain ligands were the initial scaffold used for biomimetic studies, but their macrocyclic counterparts have proven to be particularly appropriate due to their enhanced stability. Dimer and monomer complexes with such ligands have shown to be useful for a wide range of applications, which will be reviewed herein. The purpose of this review is to state with some clarity the different spectroscopic and structural characteristics of the Mn complexes formed with tetraaza macrocyclic ligands both in solution and solid-state that allow the reader to successfully identified the species involved when dealing with similar complexes of Mn.

Caffeine Targets SIRT3 to Enhance SOD2 Activity in Mitochondria.

Caffeine is chemically stable and not readily oxidized under normal physiological conditions but also has antioxidant effects, although the underlying molecular mechanism is not well understood. Superoxide dismutase (SOD) 2 is a manganese-containing enzyme located in mitochondria that protects cells against oxidative stress by scavenging reactive oxygen species (ROS). SOD2 activity is inhibited through acetylation under conditions of stress such as exposure to ultraviolet (UV) radiation. Sirtuin 3 (SIRT3) is the major mitochondrial nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which deacetylates two critical lysine residues (lysine 68 and lysine 122) on SOD2 and promotes its antioxidative activity. In this study, we investigated whether the antioxidant effect of caffeine involves modulation of SOD2 by SIRT3 using in vitro and in vivo models. The results show that caffeine interacts with SIRT3 and promotes direct binding of SIRT3 with its substrate, thereby enhancing its enzymatic activity. Mechanistically, caffeine bound to SIRT3 with high affinity (KD = 6.858 × 10–7 M); the binding affinity between SIRT3 and its substrate acetylated p53 was also 9.03 (without NAD+) or 6.87 (with NAD+) times higher in the presence of caffeine. Caffeine effectively protected skin cells from UV irradiation-induced oxidative stress. More importantly, caffeine enhanced SIRT3 activity and reduced SOD2 acetylation, thereby leading to increased SOD2 activity, which could be reversed by treatment with the SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) in vitro and in vivo. Taken together, our results show that caffeine targets SIRT3 to enhance SOD2 activity and protect skin cells from UV irradiation-induced oxidative stress. Thus, caffeine, as a small-molecule SIRT3 activator, could be a potential agent to protect human skin against UV radiation.

O-, N-Atoms-Coordinated Mn Cofactors within a Graphene Framework as Bioinspired Oxygen Reduction Reaction Electrocatalysts.

Manganese (Mn) is generally regarded as not being sufficiently active for the oxygen reduction reaction (ORR) compared to other transition metals such as Fe and Co. However, in biology, manganese-containing enzymes can catalyze oxygen-evolving reactions efficiently with a relative low onset potential. Here, atomically dispersed O and N atoms coordinated Mn active sites are incorporated within graphene frameworks to emulate both the structure and function of Mn cofactors in heme-copper oxidases superfamily. Unlike previous single-metal catalysts with general M-N-C structures, here, it is proved that a coordinated O atom can also play a significant role in tuning the intrinsic catalytic activities of transition metals. The biomimetic electrocatalyst exhibits superior performance for the ORR and zinc-air batteries under alkaline conditions, which is even better than that of commercial Pt/C. The excellent performance can be ascribed to the abundant atomically dispersed Mn cofactors in the graphene frameworks, confirmed by various characterization methods. Theoretical calculations reveal that the intrinsic catalytic activity of metal Mn can be significantly improved via changing local geometry of nearest coordinated O and N atoms. Especially, graphene frameworks containing the Mn-N3 O1 cofactor demonstrate the fastest ORR kinetics due to the tuning of the d electronic states to a reasonable state.

Dietary manganese source does not affect Mn, Zn and Cu tissue deposition and the activity of manganese-containing enzymes in lambs

Manganese (Mn) is a trace element required for normal physiological processes in animals and humans. Organic forms of trace elements are expected to have higher bioavailability in comparison with inorganic sources. The effect of feeding a diet supplemented with different sources of manganese to lambs was studied in a 112-d feeding trial. The aim of this study was to investigate the deposition of Mn in relation to activities of superoxide dismutase (SOD), manganese superoxide dismutase (MnSOD) and arginase in the tissues of lambs fed the diet supplemented with an inorganic or an organic source of manganese up to the maximum total Mn content allowed in the European Union (150mg Mn/kg). A total of eighteen female lambs of the improved Valachian breed were randomly allocated to three dietary treatments and fed an unsupplemented basal diet (Control, 31mg Mn/kg) or the identical diet supplemented with manganese sulphate (MnSO4) or manganese chelate of glycine hydrate (Mn-Gly) with a total Mn content up to 150mg/kg. Regardless of the source, feed supplementation with manganese increased Mn concentrations in plasma (P˂0.05) and the liver (P˂0.001) as well as the activity of liver MnSOD (P˂0.05) and arginase (P˂0.001) compared with the control lambs. In the kidney cortex, the concentration of Mn was greatest in lambs fed the diet supplemented with the chelated Mn source compared with animals receiving the inorganic Mn source (P˂0.05) and the unsupplemented diet (P˂0.001). The 112-d intake of feed enriched with manganese did not result in any change in Mn levels, SOD or MnSOD activity in pancreas and kidney tissues. Plasma Cu concentration was depressed in both supplemented treatments. No analyzed tissue showed a change in zinc and copper levels, except the greater Cu concentration in the liver of lambs fed the diet with Mn-Gly. The presented results did not indicate any differences between dietary Mn sources either in Mn tissue deposition or activity of SOD, MnSOD and arginase in lamb tissues; however, it seems that manganese intake of up to 150mg/kg in feed may decrease plasma Cu concentration in lambs.

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders.

Arginase in the vascular endothelium: friend or foe?

Arginase in the vascular endothelium: friend or foe?

Characterization of Monomeric MnII/III/IV–Hydroxo Complexes from X- and Q-Band Dual Mode Electron Paramagnetic Resonance (EPR) Spectroscopy

Manganese-hydroxo species have been implicated in C-H bond activation performed by metalloenzymes, but the electronic properties of many of these intermediates are not well characterized. The present work presents a detailed characterization of three Mn(n)-OH complexes (where n = II, III, and IV) of the tris[(N'-tert-butylureaylato)-N-ethylene]aminato ([H3buea](3-)) ligand using X- and Q-band dual mode electron paramagnetic resonance (EPR). Quantitative simulations for the [Mn(II)H3buea(OH)](2-) complex demonstrated the ability to characterize similar Mn(II) species commonly present in the resting states of manganese-containing enzymes. The spin states of the Mn(III) and Mn(IV) complexes determined from EPR spectroscopy are S = 2 and 3/2, respectively, as expected for the C3 symmetry imposed by the [H3buea](3-) ligand. Simulations of the spectra indicated the constant presence of two Mn(IV) species in solutions of [Mn(IV)H3buea(OH)] complex. The simulations of perpendicular- and parallel-mode EPR spectra allow determination of zero-field splitting and hyperfine parameters for all complexes. For the Mn(III) and Mn(IV) complexes, density functional theory calculations are used to determine the isotropic Mn hyperfine values, to compare the excited electronic state energies, and to give theoretical estimates of the zero-field energy.

Evaluation of plasma superoxide dismutase and glutathione peroxidase in type 2 diabetic patients

Antioxidants are agents that protect, prevent, or reduce the extent of oxidative damage to biomolecules. These agents may be enzymatic, non-enzymatic, or metal chelators. The enzymatic antioxidants include catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx). SOD, a copper, zinc and manganese-containing enzyme, reacts with superoxide radical to form hydrogen peroxide, which is then converted to water by GPx (a glutathione-dependent selenoprotein), or catalase, a heme enzyme. Decreased activity of these antioxidant enzymes may increase the susceptibility of diabetic patients to oxidative injury. An appropriate support of antioxidant supplies may help in preventing clinical complications of diabetes. In view of this, supplementary trace elements such as selenium, copper, zinc, and manganese, essential components of the enzymes, may be useful in preventing the development of diabetic complications. There are number of factors that affect an individual’s oxidative status that include gender, age, body composition, smoking status, diet, physical activity level, and the strength of defense mechanism. Hence, this study was carried out to see the relationship of these factors with antioxidant enzymes in clinically diagnosed type 2 diabetes patients.

Nucleophilic reactivity of a series of peroxomanganese(iii) complexes supported by tetradentate aminopyridyl ligands

Peroxomanganese(iii) adducts have been postulated as important intermediates in manganese-containing enzymes and small molecule oxidation catalysts. Synthetic peroxomanganese(iii) complexes are known to be nucleophilic and facilitate aldehyde deformylation, offering a convenient way to compare relative reactivities of complexes supported by different ligands. In this work, tetradentate dipyridyldiazacycloalkane ligands with systematically perturbed steric and electronic properties were used to generate a series of manganese(ii) and peroxomanganese(iii) complexes. X-Ray crystal structures of five manganese(ii) complexes all show the ligands bound to give trans complexes. Treatment of these Mn(II) precursors with H(2)O(2) and Et(3)N in MeCN at -40 °C results in the formation of peroxomanganese(iii) complexes that differ only in the identity of the pyridine ring substituent and/or the number of carbons in the diazacycloalkane backbone. To determine the effects of small ligand perturbations on the reactivity of the peroxo group, the more thermally stable peroxomanganese(iii) complexes were reacted with cyclohexanecarboxaldehyde. For these complexes, the rate of deformylation does not correlate with the expected nucleophilicity of the peroxomanganese(iii) unit, as the inclusion of methyl substituents on the pyridines affords slower deformylation rates. It is proposed that adding methyl-substituents to the pyridines, or increasing the number of carbons on the diazacycloalkane, sterically hinders nucleophilic attack of the peroxo ligand on the carbonyl carbon of the aldehyde.

Complexes of mutants of Escherichia coli aminopeptidase P and the tripeptide substrate ValProLeu

Aminopeptidase P (APPro) is a manganese-containing enzyme that catalyses the hydrolysis of the N-terminal residue of a polypeptide if the second residue is proline. Structures of APPro mutants with reduced or negligible activity have been determined in complex with the tripeptide substrate ValProLeu. In the complex of Glu383Ala APPro with ValProLeu one of the two metal sites is only partly occupied, indicating an essential role for Glu383 in metal binding in the presence of substrate. His361Ala APPro clearly possesses residual activity as the ValProLeu substrate has been cleaved in the crystals; difference electron density consistent with bound ProLeu dipeptide and a disordered Val amino acid is present at the active site. Contrary to previous suggestions, the His243Ala mutant is capable of binding substrate. The structure of the His243Ala APPro complex with ValProLeu shows that the peptide interacts with one of the active-site metal atoms via its terminal amino group. The implications of these complexes for the roles of the respective residues in APPro catalysis are discussed.

New Insights into Acetone Metabolism ▿

New Insights into Acetone Metabolism ^▿

Heterologous expression of barley and wheat oxalate oxidase in an E. coli trxB gor double mutant

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88–89% on the nucleotide level and 88–92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.

Heterologous expression of barley and wheat oxalate oxidase in an E. coli trxB gor double mutant

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88–89% on the nucleotide level and 88–92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.

Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme

Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme

Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme

Oxalate oxidase (EC 1.2.3.4) catalyses the conversion of oxalate and dioxygen into CO(2) and H(2)O(2). The barley (Hordeum vulgare) seedling root enzyme was purified to homogeneity and shown by metal analysis and EPR spectroscopy to contain Mn(II) at up to 0.80 atom per subunit. The involvement of Mn and neither flavin, Cu nor Fe in the direct conversion of dioxygen to H(2)O(2) makes oxalate oxidase unique. A model of the active site of the holoenzyme based on a homology model of the apoenzyme is proposed.

Roles of manganese and iron in the regulation of the biosynthesis of manganese-superoxide dismutase inEscherichia coli

Aerobic life-style offers both benefits and risks to living cells. The major risk comes from the formation of reactive oxygen intermediates (i.e. superoxide radical, O2-; hydrogen peroxide, H2O2; and hydroxyl radical, OH.) during normal oxygen metabolism. However, living cells are able to cope with oxygen toxicity by virtue of a unique set of antioxidant enzymes that scavenge O2- and H2O2, and prevent the formation OH.. Superoxide dismutases (SODs; EC 1.15.1.1) are metalloenzymes essential for aerobic survival. Escherichia coli contains two forms of this enzyme: an iron-containing enzyme (FeSOD) and a manganese-containing enzyme (MnSOD). In E. coli, MnSOD biosynthesis is under rigorous control. The enzyme is induced in response to a variety of environmental stress conditions including exposure to oxygen, redox cycling compounds such as paraquat which exacerbate the level of intracellular superoxide radicals, iron chelation (i.e. iron deprivation), and oxidants. A model for the regulation of the MnSOD has been proposed in which the MnSOD gene (sodA) is negatively regulated at the level of transcription by an iron-containing redox-sensitive repressor protein. The effect of iron-chelation most probably results in removal of the iron necessary for repressor activity. Recent studies have shown that sodA expression is regulated by three iron-dependent regulatory proteins, Fur (ferric uptake regulation), Fnr (fumarate nitrate regulation) and SoxR (superoxide regulon), and by the ArcA/ArcB (aerobic respiration control) system. The potential Fur-, Fnr- and ArcA-binding sites in the sodA promoter region have been identified by using different cis-acting regulatory mutations that caused anaerobic derepression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide Dismutase Gene Expression in the Endotoxin-Treated Rat Lung

Background It is well known that oxygen free radicals (OFR) play a vital role in the various type of acute lung injury. Among various antioxidant defense mechanisms, the superoxide dismutases (SOD) are thought to be the first line of antioxidant defense by catalyzing the dismutation of two superoxide radicals to yield hydrogen peroxide and oxygen. Eukaryotic cells contain two types of intracellular SOD : cytosolic, dimeric copper/zinc- containing enzyme (CuZnSOD) and mitochondrial, tetrameric manganese-containing enzyme (MnSOD). The purpose of this study is to evaluate the time-dependent gene expression of MnSOD and CuZnSOD in the endotoxin-treated rats, and to compare with the manifestations of LPS-induced acute lung injury in rats. Methods Total RNA from rat lung was isolated using single step phenol extraction 0, 1, 2, 4, 6, 12, 18, 24 hours after E. coli endotoxin injection (n=3, respectively). RNA was separated by formaldehyde-containing 1.2% agarose gels elctrophoresis, transblotted, baked, prehybridized, and hybridized with 32P-labeled cDNA probes for rat MnSOD and CuZnSOD, which were kindly donated by Dr. Ho (Duke University, Durham, NC, USA). The probes were labeled by nick translation. Blots were washed and autoradiography were quantitated using laser densitometry. Equivalent amounts of total RNA/gel were assessed by monitoring 285 and 185 rRNA. Results Endotoxin caused a rise in steady-state MnSOD mRNA levels by 4h with peak mRNA accumulation by 6h. Continued MnSOD mRNA expression was observed at 12h. CuZnSOD mRNA expression was observed from 1h to 24h with peak levels by 18h. Conclusion These results suggest that SOD palys an important defensive role in the endotoxin-induced acute lung injury in rats.

Evidence that the alkaline P-nitrophenylphosphate phosphatase from Halobacterium halobium is a manganese-containing enzyme

1. 1. Alkaline p-nitrophenylphosphate phosphatase of Halobacterium halobiium, either purified or in crude extracts, was progressively inactivated by treatment with several metal chelators. 2. 2. The activity of treated crude extracts was fully restored in the presence of 25–50 μM Mn 2+ or 1 mM Co 2+, and partially restored in the presence of 1 mM Cd 2+. 3. 3. Zn 2+ ions, as well as other divalent cations tested, were without effect. 4. 4. In the presence of a saturating concentration of Mn 2+, but not Co 2+ or Cd 2+, the activity of the metal-depleted enzyme reached values well over the native control activity. 5. 5. Activation of the metal-depleted enzyme by Mn 2+ showed cooperative kinetics, whereas activation by Co 2+ showed Lineweaver-Burk kinetics. 6. 6. The results suggest that the enzyme contains two different types of metal-binding sites: essential site(s), occupied by endogenous Mn 2+ ions, and regulatory site(s), that can be occupied by exogenous Mn 2+ with an activating effect.

Antioxidant defenses in the ground squirrel Citellus citellus 2. The effect of hibernation

In spring and autumn, the ground squirrel Citellus citellus is awake and active but in winter it usually hibernates. Reawakening from hibernation involves intense metabolic activity in the interscapular brown adipose tissue (IBAT). The IBAT of hibernation animals showed significant increases in the activities of superoxide dismutase (both copper-zinc and manganese-containing enzymes), glutathione peroxidase, and in the amount of ascorbate present. Glutathione peroxidase also increased in the liver, as did ascorbate in the plasma. These changes were not merely a consequence of exposure to low environmental temperatures. It is proposed that antioxidant defenses are increased in the IBAT of ground squirrel at the onset of hibernation in order to protect the tissue from reactive oxygen species generated as a result of the intense metabolic activity sustained by this tissue during reawakening.

Manganese-containing superoxide-dismutase deficiency in polymorphonuclear leukocytes of adults with rheumatoid arthritis.

Superoxide dismutase (SOD) is known to regulate the level of superoxide radicals inside cells. The purpose of this work was to investigate the role of SOD activity in tissue damage produced by superoxide radicals. SOD was measured in polymorphonuclear cells of patients with rheumatoid arthritis and controls. The distinct SOD activities, including manganese-containing and copper-zinc-containing enzymes, were evaluated in cytoplasma and mitochondria of human granulocytes. Except for the comparison between total SOD and cytoplasmic copper-zinc SOD, no correlation was found among the different SOD levels. Moreover, a significant decrease was observed only for cytoplasmic manganese-containing enzyme in granulocytes of adults with rheumatoid arthritis. These data confirm the necessity of evaluation of various SOD classes and suggest the interest of biochemical tests in granulocytes for early diagnosis and better comprehension of tissue damage due to inflammation.