Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: An unexpected role for intracellular chloride channels

GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel. gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu+ to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl−. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl− there is very little Cu-loading at any pH. Cl− has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl− may also bind to Fet3p and that Cu+ has an allosteric effect on the binding of Cl− to the enzyme. Thus, Cl− may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.

Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase.

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

Spectroscopy of selected copper group minerals : chalcophyllite and chenevixite-implications for hydrogen bonding

NIR and IR spectroscopy has been applied for detection of chemical species and the nature of hydrogen bonding in arsenate complexes. The structure and spectral properties of copper(II) arsenate minerals chalcophyllite and chenevixite are compared with copper(II) sulphate minerals devilline, chalcoalumite and caledonite. Split NIR bands in the electronic spectrum of two ranges 11700-8500 cm-1 and 8500-7200 cm-1 confirm distortion of octahedral symmetry for Cu(II) in the arsenate complexes. The observed bands with maxima at 9860 and 7750 cm-1 are assigned to Cu(II) transitions 2B1g ® 2B2g and 2B1g ® 2A1g. Overlapping bands in the NIR region 4500-4000 cm-1 is the effect of multi anions OH-, (AsO4)3- and (SO4)2-. The observation of broad and diffuse bands in the range 3700-2900 cm-1 confirms strong hydrogen bonding in chalcophyllite relative to chenevixite. The position of the water bending vibrations indicates the water is strongly hydrogen bonded in the mineral structure. The strong absorption feature centred at 1644 cm-1 in chalcophyllite indicates water is strongly hydrogen bonded in the mineral structure. The H2O-bending vibrations shift to low wavenumbers in chenevixite and an additional band observed at 1390 cm-1 is related to carbonate impurity. The characterisation of IR spectra by ν3 antisymmetric stretching vibrations of (SO4)2- and (AsO4)3 ions near 1100 and 800 cm-1 respectively is the result of isomorphic substitution for arsenate by sulphate in both the minerals of chalcophyllite and chenevixite.

Raman microscopy study of tyrolite : a multi-anion arsenate mineral

The Raman spectrum of tyrolite, CaCu5(AsO4)2(CO3)(OH) 4.6H2O, from Brixlegg, Tyrol, Austria, is reported. Comparison with copper hydroxy-arsenate and basic carbonates was used to achieve assignments of the observed bands. The AsO43- group is characterized by two υ4 modes around 433 and 480 cm-1 plus a broad band around 840 cm-1 as the υ overlapping with the υ. The υ3 mode is observed as a single band around 355 cm -1. The CO32- υ1 mode is observed around 1035 and 1088 cm-1, although this assignment is difficult because of the in-plane OH bending vibrations at similar frequencies. Two υ4 modes are assigned to the 717 and 755 cm-1 bands. The υ3 mode is present as three bands at 1431, 1463, and 1498 cm-1. A large split caused by bridging carbonates may explain the band at 1370 cm -1. The H2O bending region shows two bands at 1635 and 1667 cm-1 together with stretching modes around 3204 and 3303 cm-1, the first associated with adsorbed H2O, while the second indicates more strongly bonded H2O. Three bands around 3534, 3438, and 3379 cm -1 are assigned to OH stretching modes of the OH groups in the crystal structure. The 202, 262, 301, 524, and 534 cm-1 bands are assigned to Cu-OH bending and stretching modes, whereas the bands around 179, 202, and 217 cm-1 are ascribed to O-(Ca, Cu)-O(H) with the O(H) at much greater distance from the cation. The bands around 503, 570, and 598 cm-1 are ascribed to the Cu-O stretching modes.

Raman spectroscopy of the multi anion mineral arsentsumebite Pb2Cu(AsO4)(SO4)(OH) and in comparison with tsumebite Pb2Cu(PO4)(SO4)(OH)

The mineral arsentsumebite Pb2Cu(AsO4)(SO4)(OH), a copper arsenate-sulfate hydroxide of the brackebuschite group has been characterised by Raman spectroscopy. The brackebuschite mineral group are a series of monoclinic arsenates, phosphates and vanadates of the general formula A2B(XO4)(OH,H2O), where A may be Ba, Ca, Pb, Sr, while B may be Al, Cu2+,Fe2+, Fe3+, Mn2+, Mn3+, Zn and XO4 may be AsO4, PO4, SO4,VO4. Bands are assigned to the stretching and bending modes of SO42- AsO43- and HOAsO3 units. Raman spectroscopy readily distinguishes between the two minerals arsentsumebite and tsumebite. Raman bands attributed to arsenate are not observed in the Raman spectrum of tsumebite. Phosphate bands found in the Raman spectrum of tsumebite are not found in the Raman spectrum of arsentsumebite. Raman spectroscopy readily distinguishes the two minerals tsumebite and arsentsumebite.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Anthranilic acid oxidase system of Tecoma stans—II. : Studies on the properties of a purified anthranilic acid oxidase system and its separation into different enzymic components

An enzyme system which catalysed the conversion of anthranilic acid to catechol has been purified 20-fold from a cell-free leaf extract of Tecoma stans. The optimum substrate concentration was 10−3 M and optimum temperature for the reaction was 45°. The presence of a multi-enzyme system was inferred from inhibition studies. The formation of catechol was inhibited by Mg2+, Zn2+, and Co2+ ions, whereas anthranilic acid disappearance was not affected to the same extent. The effect of metal chelating agents like EDTA, cyanide and pyrophosphate showed a similar trend. PCMB inhibited catechol formation but had no effect on anthranilic acid disappearance. The reaction was not inhibited by catalase, nor was it activated by peroxide-donating systems. This ruled out the possibility of peroxidative type of reaction. The overall reaction is markedly activated by NADPH and THFA. This multi-enzyme was separated into three different components, by fractionation with Alumina Cγ and calcium phosphate gels. The overall reaction catalysed by these components can be represented as anthranilic acid→3-hydroxy anthranilic acid→o-aminophenol→catechol.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Synthesis, Crystal Structure, and Magnetic Properties of a Ferromagnetically Coupled Angular Trinuclear Copper(11) Complex [Cu3(02CMe)4(bpy)s(HzO)](PF)62

The synthesis, X-ray crystal structure, and magnetic properties of an angular trinuclear copper(II) complex [Cu3(O2CMC)4(bpy)3(H2O)](PF6)2 (1), obtained from a reaction of Cu2(O2CMe)4(H2O)2 With 2,2'-bipyridine (bpy) and NH4PF6 in ethanol, are reported. Complex 1 crystallizes in triclinic space group P1BAR with a = 11.529(1) angstrom, b = 12.121(2) angstrom, c = 17.153(2) angstrom, alpha = 82.01(1)-degrees, beta = 79.42(1)-degrees, gamma = 89.62(1)-degrees, and Z = 2. A total of 6928 data with I > 2.5sigma(I) were refined to R = 0.0441 and R(w) = 0.0557. The structure consists of a trinuclear core bridged by four acetate ligands showing different bonding modes. The coordination geometry at each copper is distorted square-pyramidal with a CuN2O2...O chromophore. The Cu...Cu distances are 3.198(1) angstrom, 4.568(1) angstrom, and 6.277(1) angstrom. There are two monoatomic acetate bridges showing Cu-O-Cu angles of 93.1(1) and 97.5(1)-degrees. Magnetic studies in the temperature range 39-297 K show the presence of a strong ferromagnetically coupled dicopper(II) unit (2J = +158 cm-1) and an essentially isolated copper(II) center (2J' = -0.4 cm-1) in 1. The EPR spectra display an axial spectrum giving g(parallel-to) = 2.28 (A(parallel-to) = 160 X 10(-4) cm-1) and g(perpendicular-to) = 2.06 (A(perpendicular-to) = 12 X 10(-4) cm-1) for the normal copper and two intense isotropic signals with g values 2.70 and 1.74 for the strongly coupled copper pair. The structural features of 1 compare well with the first generation models for ascorbate oxidase.

Studies on multi-metal ion tolerance of Thiobacillus ferrooxidans

The influence of different concentrations of base metal ions, such as CU2+, Zn2+ and Fe3+, when present either alone or in different possible binary and ternary combinations in a 9K medium, on the fel rous ion oxidation ability of Thiobacillus ferrooxidans was studied. Levels and degree of toxicity of these ions have been quantified in terms of toxicity index (TI). Copper and zinc tolerant strains of the bacteria were developed through serial subculturing and their activity tested in the presence of the above metal ions in comparison with the behavior of wild unadapted cells under similar conditions. Copper tolerant strains (25 g/L Cu2+) were found to be more efficient in the bioleaching of both copper and zinc concentrates than wild unadapted strains, while zinc tolerant strains (40 g/L Zn2+) exhibited better leaching efficiency only in the bioleaching of sphalerite concentrates. The significance and relevance of multi-metal ion tolerance in Thiobacillus ferrooxidans has been highlighted with respect to bioleaching of sulphide mineral concentrates. (C) 1997 Published by Elsevier Science Ltd.