Expression and functional analysis of glutamate synthase small subunit-like proteins from archaeon Pyrococcus horikoshii

Glutamate synthase, glutamine α-ketoglutarate amidotransferase (often abbreviated as GOGAT) is a key enzyme in the early stages of ammonia assimilation in bacteria, algae and plants, catalyzing the reductive transamidation of the amido nitrogen from glutamine to α-ketoglutarate to form two molecules of glutamate. Most bacterial glutamate synthases consist of a large and small subunit. The genomes of three Pyrococcus species harbour several open reading frames which show homology with the small subunit of glutamate synthase. There are no open reading frames which may be coding for a large subunit responsible for the glutamate formation in these pyrococcal genomes.In this work, two open reading frames PH0876 and PH1873 from P. horikoshii were cloned and expressed in Escherichia coli as soluble proteins. Both proteins show NADPH-dependent oxidoreductase activity using artificial electron acceptors iodonitrotetrazolium chloride at thermophilic conditions. It is possible that these open reading frames are the products of gene duplication and that they are the early forms of an electron transfer domain in archaea which may have later contributed to many electron transfer enzymes.     

Bioinspired FeIIICdII and FeIIIHgII complexes: synthesis, characterization and promiscuous catalytic activity evaluation

In this work we report on the synthesis, crystal structure, and physicochemical characterization of the novel dinuclear [Fe^IIICd^II(L)(μ-OAc)₂]ClO₄·0.5H₂O (1) complex containing the unsymmetrical ligand H₂L = 2-bis[{(2-pyridyl-methyl)-aminomethyl}-6-{(2-hydroxy-benzyl)-(2-pyridyl-methyl)}-aminomethyl]-4-methylphenol. Also, with this ligand, the tetranuclear [Fe₂^IIIHg₂^II(L)₂(OH)₂](ClO₄)₂·2CH₃OH (2) and [Fe^IIIHg^II(L)(μ-CO₃)Fe^IIIHg^II(L)](ClO₄)₂·H₂O (3) complexes were synthesized and fully characterized. It is demonstrated that the precursor [Fe^III₂Hg^II₂(L)₂(OH)₂](ClO₄)₂·2CH₃OH (2) can be converted to (3) by the fixation of atmospheric CO₂ since the crystal structure of the tetranuclear organometallic complex [Fe^IIIHg^II(L)(μ-CO₃)Fe^IIIHg^II(L)](ClO₄)₂·H₂O (3) with an unprecedented {Fe^III(μ-O_phenoxo)₂(μ-CO₃)Fe^III} core was obtained through X-ray crystallography. In the reaction 2 → 3 a nucleophilic attack of a Fe^III-bound hydroxo group on the CO₂ molecule is proposed. In addition, it is also demonstrated that complex (3) can regenerate complex (2) in aqueous/MeOH/NaOH solution. Magnetochemical studies reveal that the Fe^III centers in 3 are antiferromagnetically coupled (J = − 7.2 cm^− 1) and that the Fe^III-OR-Fe^III angle has no noticeable influence in the exchange coupling. Phosphatase-like activity studies in the hydrolysis of the model substrate bis(2,4-dinitrophenyl) phosphate (2,4-bdnpp) by 1 and 2 show Michaelis-Menten behavior with 1 being ~ 2.5 times more active than 2. In combination with k_H/k_D isotope effects, the kinetic studies suggest a mechanism in which a terminal Fe^III-bound hydroxide is the hydrolysis-initiating nucleophilic catalyst for 1 and 2. Based on the crystal structures of 1 and 3, it is assumed that the relatively long Fe^III…Hg^II distance could be responsible for the lower catalytic effectiveness of 2.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Desacetoxycephalosporin C synthetase: Importance of order of cofactor/ reactant addition

The ring expansion enzyme (desacetoxycephalosporin C synthetase) of cephalosporin C biosynthesis in Cephalosporium acremonium has been purified 40-fold, using gel filtration on LKB Ultrogel AcA54. Purity was about twice that previously attained. The purified enzyme showed an almost absolute requirement for α-ketoglutarate, and most other acids were inactive or only weakly active. The order of addition of the cofactors and reactants was found to exert a major effect on enzyme activity. The major negative effect was caused by α-ketoglutarate or penicillin N being added to the enzyme first. The most effective order of addition to the enzyme was found to be the simultaneous addition of Fe²⁺, ascorbate and ATP, followed by α-ketoglutarate and then penicillin N. The negative effect of the required reactant, α-ketoglutarate, when added too early, coincides with observations made about other α-ketoglutarate-dependent dioxygenases even though, in the case of the synthetase, one atom of oxygen does not end up in the product, desacetoxycephalosporin C.     

Reaction of haem containing proteins and enzymes with hydroperoxides: The radical view

The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe³⁺ (ferric) state reacts with hydroperoxides with a formation of the Fe⁴⁺=O (oxoferryl) haem state and a free radical primarily located on the π-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; αTyr42, αTrp14, βTrp15, βCys93, (αTyr24−αHis20), all in the α- and β-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases.     

Thymine 7-hydroxylase activity in normal and leukemic leukocytes

Thymine 7-hydroxylase activity was detected in normal and leukemic leukocytes of Fischer 344 rats as witnessed by the conversion of thymine-2-¹⁴C to 5-hydroxymethyluracil. All the enzyme activity was found inside the cells, but occasionally low levels of activity were recovered from the plasma. The catalysis of the reaction was stimulated by ascorbic acid, α-ketoglutaric acid and Fe²⁺. Leukemic cells produced up to eight times as much product as normal leukocytes.     

Synthesis, characterization and antibacterial activity of Fe, Co, Cu and Zn complexes probed by transmission electron microscopy

We synthesized iron(III), cobalt(II), copper(II) and zinc(II) complexes [Fe^III(HBPClNOL)Cl₂]·H₂O (1), [Co^II(H₂BPClNOL)Cl₂] (2), [Cu^II(H₂BPClNOL)Cl]Cl·H₂O (3), and [Zn^II(HBPClNOL)Cl] (4), where H₂BPClNOL is the ligand (N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)[(3-chloro)(2-hydroxy)]propylamine). The complexes obtained were characterized by elemental analysis, IR and UV-visible spectroscopies, electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and cyclic voltammetry. X-ray diffraction studies were performed for complexes (3) and (4) revealing the presence of mononuclear and dinuclear structures in solid state for (3). However, the zinc complex is mononuclear in solid state. Biological studies of complexes (1)-(4) were carried out in vitro for antimicrobial activity against nine Gram-positive bacteria (Staphylococcus aureus strains RN 6390B, COL, ATCC 25923, Smith Diffuse, Wood 46, enterotoxigenic S. aureus FRI-100 (SEA+), FRI S-6 (SEB+) and SEC FRI-361) and animal strain S. aureus LSA 88 (SEC/SED/TSST-1+). The following sequence of inhibition promoted by the complexes was observed: (4) > (2) > (3) > (1), showing the effect of the metal on the biological activity. To directly observe the morphological changes of the internal structure of bacterial cells after the treatment, transmission electron microscopy (TEM) was employed. For the most active complex [Zn^II(HBPClNOL)Cl] (4), granulation deposits around the genetic material and internal material leaking were clearly detected.     

Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis

Cell-free preparations from Rhodotorula glutinis catalyzed the conversion of deoxyribonucleosides to ribonucleosides in a pyrimidine deoxyribonucleoside 2' -hydroxylase reaction. The reaction occurred with only thymidine or deoxyuridine, of the common deoxyribonucleosides, without detachment of the deoxyribose moiety, at the nucleoside level. The same enzyme preparations catalyzed the conversion of thymine to 5-hydroxymethyluracil in a thymine 7-hydroxylase reaction. Requirements for molecular oxygen, alpha-ketoglutarate, Fe2+, and ascorbate indicated that the 2' -hydroxylase and 7-hydroxylase reactions are of the alpha-keto-acid dioxygenases class. The requirements for alpha-ketoglutarate and Fe2+ were very stringent. During the course of the 2' -hydroxylase and 7-hydroxylase reactions, alpha-ketoglutarate was decarboxylated to form succinate and CO2 so that the ratio of hydroxylated nucleoside or pyrimidine to CO2 was 1:1.5-Hydroxymethyluracil and 5-formyluracil also stimulated the decarboxylation of alpha-ketoglutarate and thus appeared to undergo 7-hydroxylase reactions.     

Regulation of pyrimidine salvage in Aspergillus nidulans: a role for the major regulatory gene are A mediating nitrogen metabolite repression

Summary The synthesis of thymine 7-hydroxylase, an -ketoglutarate dependent dioxygenase, is subject both to nitrogen metabolite repression and to oxygen repression, while synthesis of the other pyrimidine salvage pathway dioxygenase, pyrimidine deoxyribonucleoside 2-hydroxylase, is subject to neither. areA300, an allele of the positive acting regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans, considerably elevates levels of thymine 7-hydroxylase, probably alleviating at least partly both nitrogen metabolite repression and oxygen repression. areA300 has little or no effect on levels of pyrimidine deoxyribonucleoside 2-hydroxylase but does elevate net uptake capacities for thymine, thymidine and deoxyuridine two-fold. areA300 was selected as allowing thymine to supplement a pyrimidine auxotrophy and was found to allow supplementation by thymidine, other pyrimidine nucleosides and pyrimidine salvage intermediates as well. This is the first reported evidence for areA control over an activity(-ies) not directly concerned with nitrogen source utilization.     

Spectroscopic, catalytic and binding properties of Bacillus subtilis NO synthase-like protein: comparison with other bacterial and mammalian NO synthases

Genome sequencing has shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles and properties of these proteins remain unclear. UV-visible spectroscopy was used to characterize the recombinant NOS-like protein from Bacillus subtilis (bsNOS) in its ferric and ferrous states in the presence of various Fe^III- and Fe^II-heme-ligands and of a series of l-arginine (l-arg) analogs. BsNOS exhibited several spectroscopic and binding properties in common with Bacillus anthracis NOS (baNOS) that were clearly different from those of tetrahydrobiopterin (H4B)-free mammalian NOS oxygenase domains (mNOS_oxys) and of Staphylococcus aureus NOS (saNOS). Interestingly, bsNOS and baNOS that do not contain H4B exhibited properties much closer to those of H4B-containing mNOS_oxys. Moreover, bsNOS was found to efficiently catalyze the oxidation of l-arginine into l-citrulline by H₂O₂, whereas H4B-free mNOS_oxys exhibited low activities for this reaction.     

Radically different amyloid conformations dictate the seeding specificity of a chimeric Sup35 prion

PHF2 belongs to a class of α-ketoglutarate-Fe²⁺-dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD, binds Lys4-trimethylated histone 3 in submicromolar affinity and has been reported to have the demethylase activity of monomethylated lysine 9 of histone 3 in vivo. However, we did not detect demethylase activity for PHF2 Jumonji domain (with and without its linked PHD) in the context of histone peptides. We determined the crystal structures of PHF2 Jumonji domain in the absence and presence of additional exogenous metal ions. When Fe²⁺ or Ni²⁺ was added at a high concentration (50 mM) and allowed to soak in the preformed crystals, Fe²⁺ or Ni²⁺ was bound by six ligands in an octahedral coordination. The side chains of H249 and D251 and the two oxygen atoms of N-oxalylglycine (an analog of α-ketoglutarate) provide four coordinations in the equatorial plane, while the hydroxyl oxygen atom of Y321 and one water molecule provide the two axial coordinations as the fifth and sixth ligands, respectively. The metal binding site in PHF2 closely resembles the Fe²⁺ sites in other Jumonji domains examined, with one important difference—a tyrosine (Y321 of PHF2) replaces histidine as the fifth ligand. However, neither Y321H mutation nor high metal concentration renders PHF2 an active demethylase on histone peptides. Wild type and Y321H mutant bind Ni²⁺ with an approximately equal affinity of 50 μM. We propose that there must be other regulatory factors required for the enzymatic activity of PHF2 in vivo or that perhaps PHF2 acts on non-histone substrates. Furthermore, PHF2 shares significant sequence homology throughout the entire region, including the above-mentioned tyrosine at the corresponding iron-binding position, with that of Schizosaccharomyces pombe Epe1, which plays an essential role in heterochromatin function but has no known enzymatic activity.     

Cloning,heterologous expression,and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris

The cytochrome (cyt) c′, cyt c₅₅₆, and cyt c₂ genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c′ and cyt c₅₅₆ have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b₅₆₂, in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe^II and Fe^III states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe^II-cyt c₅₅₆ is more stable than the corresponding state of Fe^III-cyt c₅₅₆ (ΔΔG_f°=22 kJ/mol).     

Coordination diversity of N-phosphoryl-N′-phenylthiourea (LH) towards Co, Ni and Pd cations: Crystal structure of ML2-N,S and ML2-O,S chelates

Thiourea, PhNHC(S)NHP(O)(OPrⁱ)₂ (LH) chelates of Co^II, Ni^II, and Pd^II ions have been obtained and investigated by single-crystal X-ray diffraction, UV, IR, NMR spectroscopy, and EI mass-spectrometry. The unusual 1,3-N,S-coordination via sulfur and NP(O) nitrogen atoms has been found in the trans-square-planar NiL₂ and PdL₂ complexes, whereas the 1,5-O,S-coordination is realized in the tetrahedral CoL₂ complex. DFT calculations have revealed significant stabilization of the 1,3-N,S-structures due to stronger crystal field and the NH-OP hydrogen bonds.     

Substrate entasis and electronic coupling elements in electron transfer from Fe in a multicopper ferroxidase

Outersphere electron transfer in multicopper oxidases occurs at the type 1, blue Cu^II. One class of MCO proteins exhibits a specificity in this reaction towards Fe^II. In work carried out in collaboration with the Solomon lab over the past 7 years, we have delineated the structural motifs that support this ferroxidase specificity and have quantified the contributions that each makes to this outersphere electron transfer reaction from Fe^II to the type 1 Cu^II. Two features of this electron transfer catalysis stand out. First, the protein provides a binding site for Fe^II that actually favors Fe^III; this coordination sphere places the bound Fe^II in a state of “entasis” that can be relieved by loss of an electron. In short, the E^° of the bound Fe^II is lowered relative to that of aqueous ferrous iron making electron transfer thermodynamically favorable. Second, carboxylates within this coordination sphere provide an electronic coupling pathway for the electron transfer via their H-bond network with type 1 Cu histidine ligands thus making electron transfer kinetically efficient. This brief report breaks down these contributions to ferroxidase specificity in terms of the semi-classical Marcus equation describing outersphere electron transfer.     

N-NMR characterization of His residues in and around the active site of FeSOD

We have exploited ¹⁵N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His′, paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His′ that ligate Fe^II. These confirm that neither the Q69H nor the Q69E mutation strongly affects the Fe^II electronic structure, despite the 250 mV and > 660 mV increases in E_m they produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to Fe^II, as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to Fe^IISOD outside the Fe^II coordination sphere, affecting Gln69 but without direct involvement of His30.     

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe^IIICu^II(BPBPMP)(OAc)₂]ClO₄, 1, with the unsymmetrical N₅O₂ donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H₂BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe^III–()OH–Cu^IIOH₂]⁺ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe^III-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe^IIIM^II, where M^II is Zn^II, Mn^II, Ni^II, or Fe^II). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 M), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10⁷ over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.Electronic Supplementary Material Supplementary material is available for this article at .     

Investigation of iron pools in cucumber roots by Mössbauer spectroscopy: direct evidence for the Strategy I iron uptake mechanism

Distinct chemical species of iron were investigated by Mössbauer spectroscopy during iron uptake into cucumber roots grown in unbuffered nutrient solution with or without ⁵⁷Fe-citrate. Mössbauer spectra of iron deficient roots supplied with 10–500 μM ⁵⁷Fe-citrate for 30–180 min and 24 h and iron-sufficient ones, were recorded. The roots were analysed for Fe concentration and Fe reductase activity. The Mössbauer parameters in the case of iron-sufficient roots revealed high-spin iron(III) components suggesting the presence of Fe^III-carboxylate complexes, hydrous ferric oxides and sulfate–hydroxide containing species. No Fe^II was detected in these roots. However, iron-deficient roots supplied with 0.5 mM ⁵⁷Fe^III-citrate for 30 min contained significant amount of Fe^II in a hexaaqua complex form. This is a direct evidence for the Strategy I iron uptake mechanism. Correlation was found between the decrease in Fe reductase activity and the ratio of Fe^II–Fe^III components as the time of iron supply was increased. The data may refer to a higher iron reduction rate as compared to its uptake/reoxidation in the cytoplasm in accordance with the increased reduction rate in iron deficient Strategy I plants.     

Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

The template synthesis of dimetallic complexes

The Fe^II ion acts as a template to generate a dinuclear triple-stranded complex, in which two tris-diimine compartments are separated by rigid diphenylurea spacers. The template reaction involves the combination of 11 particles and leads to the formation of a single highly symmetrical product, as shown by X-ray diffraction studies. The diiron(II) complex undergoes reversible oxidation to the Fe^III derivative. On the other hand, the Cu^I centre promotes the template formation of a double stranded dinuclear complex, which shows a total and unique resistance to the oxidation to Cu^II. Such an intriguing feature results (i) from the bulkiness of the substituents, which hinders the planarization of the donor set, and (ii) from the rigidity of the diphenylurea spacers, which prevent disassembling of the double stranded complex and formation of two mononuclear chelated Cu^I species.