RESUMO
The importance of the vitamin B6-derived pyridoxal cofactor for human health has been established through more than 70 years of intensive biochemical research, revealing its fundamental roles in metabolism. B6 deficiency, resulting from nutritional limitation or impaired uptake from dietary sources, is associated with epilepsy, neuromuscular disease and neurodegeneration. Hereditary disorders of B6 processing are also known, and genetic defects in pathways involved in transport of B6 into the cell and its transformation to the pyridoxal-5'-phosphate enzyme cofactor can contribute to cardiovascular disease by interfering with homocysteine metabolism and the biosynthesis of vasomodulatory polyamines. Compared to the processes involved in cellular uptake and processing of the B6 vitamers, trafficking of the PLP cofactor across intracellular membranes is very poorly understood, even though the availability of PLP within subcellular compartments (particularly the mitochondrion) may have important health implications. The aim of this review is to concisely summarize the state of current knowledge of intracellular trafficking of PLP and to identify key directions for future research.
Assuntos
Glicogênio/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Fosfato de Piridoxal/metabolismo , Frações Subcelulares/metabolismo , Animais , Membrana Celular , Humanos , Transporte Proteico/fisiologiaRESUMO
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction takes place at the tetra-nuclear manganese calcium-oxo (Mn4Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn4Ca-oxo cluster and its environment in the S2 state of the OEC. The assignments for the electron-nuclear magnetic interactions that were observed in these studies were facilitated by the use of synthetic dimanganese di-µ-oxo complexes. However, there is an immense need to understand the effects of the protein environment on the coordination geometry of the Mn4Ca-oxo cluster in the OEC of PSII. In the present study, we use a proteinaceous model system to examine the protein ligands that are coordinated to the dimanganese catalytic center of manganese catalase from Lactobacillus plantarum. We utilize two-dimensional hyperfine sublevel correlation (2D HYSCORE) spectroscopy to detect the weak magnetic interactions of the paramagnetic dinuclear manganese catalytic center of superoxidized manganese catalase with the nitrogen and proton atoms of the surrounding protein environment. We obtain a complete set of hyperfine interaction parameters for the protons of a water molecule that is directly coordinated to the dinuclear manganese center. We also obtain a complete set of hyperfine and quadrupolar interaction parameters for two histidine ligands as well as a coordinated azide ligand, in azide-treated superoxidized manganese catalase. On the basis of the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multinuclear manganese clusters.
Assuntos
Catalase/química , Complexo de Proteína do Fotossistema II/química , Superóxidos/química , Azidas/química , Lactobacillus plantarum , Fenômenos Magnéticos , Modelos Químicos , Nitrogênio/química , Prótons , Análise Espectral , Água/químicaRESUMO
Biochemical communication between the cytoplasmic and mitochondrial subsystems of the cell depends on solute carriers in the mitochondrial inner membrane that transport metabolites between the two compartments. We have expressed and purified a yeast mitochondrial carrier protein (Mtm1p, YGR257cp), originally identified as a manganese ion carrier, for biochemical characterization aimed at resolving its function. High affinity, stoichiometric pyridoxal 5'-phosphate (PLP) cofactor binding was characterized by fluorescence titration and calorimetry, and the biochemical effects of mtm1 gene deletion on yeast mitochondria were investigated. The PLP status of the mitochondrial proteome (the mitochondrial 'PLP-ome') was probed by immunoblot analysis of mitochondria isolated from wild type (MTM1(+)) and knockout (MTM1(-)) yeast, revealing depletion of mitochondrial PLP in the latter. A direct activity assay of the enzyme catalyzing the first committed step of heme biosynthesis, the PLP-dependent mitochondrial enzyme 5-aminolevulinate synthase, extends these results, providing a specific example of PLP cofactor limitation. Together, these experiments support a role for Mtm1p in mitochondrial PLP trafficking and highlight the link between PLP cofactor transport and iron metabolism, a remarkable illustration of metabolic integration.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfato de Piridoxal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Proteínas de Transporte/genética , Deleção de Genes , Ferro/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The Saccharomyces cerevisiae mitochondrial carrier YGR257Cp (Mtm1p) is an integral membrane protein that plays an essential role in mitochondrial iron homeostasis and respiratory functions, but its carrier substrate has not previously been identified. Large amounts of pure protein are required for biochemical characterization, including substrate screening. Functional complementation of a Saccharomyces knockout by expression of TwinStrep tagged YGR257Cp demonstrates that an affinity tag does not interfere with protein function, but the expression level is very low. Heterologous expression in Pichia pastoris improves the yield but the product is heterogeneous. Expression has been screened in several Escherichia coli hosts, optimizing yield by modifying induction conditions and supplementing with rare tRNAs to overcome codon bias in the eukaryotic gene. Detection of an additional N-terminal truncation product in E. coli reveals the presence of a secondary intracistronic translation initiation site, which can be eliminated by silent mutagenesis of an alternative (Leu) initiation codon, resulting in production of a single, full-length polypeptide (â¼30% of the total protein) as insoluble inclusion bodies. Purified inclusion bodies were successfully refolded and affinity purified, yielding approximately 40mg of pure, soluble product per liter of culture. Refolded YGR257Cp binds pyridoxal 5'-phosphate tightly (KD<1µM), supporting a new hypothesis that the mitochondrial carrier YGR237Cp and its homologs function as high affinity PLP transporters in mitochondria, providing the first evidence for this essential transport function in eukaryotes.
RESUMO
Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.
Assuntos
Sistema Livre de Células , Biossíntese de Proteínas , Biotecnologia/métodos , Biologia Molecular/métodos , Tecnologia Farmacêutica/métodosRESUMO
Human manganese superoxide dismutase (Sod2p) has been expressed in yeast and the protein purified from isolated yeast mitochondria, yielding both the metallated protein and the less stable apoprotein in a single chromatographic step. At 30 °C growth temperature, more than half of the purified enzyme is apoprotein that can be fully activated following reconstitution, while the remainder contains a mixture of manganese and iron. In contrast, only fully metallated enzyme was isolated from a similarly constructed yeast strain expressing the homologous yeast manganese superoxide dismutase. Both the manganese content and superoxide dismutase activity of the recombinant human enzyme increased with increasing growth temperatures. The dependence of in vivo metallation state on growth temperature resembles the in vitro thermal activation behavior of human manganese superoxide dismutase observed in previous studies. Partially metallated human superoxide dismutase is fully active in protecting yeast against superoxide stress produced by addition of paraquat to the growth medium. However, a splice variant of human manganese superoxide dismutase (isoform B) is expressed as insoluble protein in both Escherichia coli and yeast mitochondria and did not protect yeast against superoxide stress.
Assuntos
Metais/metabolismo , Saccharomyces cerevisiae/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Transporte Biológico , Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/química , Superóxidos/metabolismo , TemperaturaRESUMO
The synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (OEC) of photosystem II (PSII). Understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the OEC. Mn catalase in its Mn(III)-Mn(IV) state is a protein model of the OEC's S(2) state. From (17)O-labeled water exchanged into the di-µ-oxo di-Mn(III,IV) coordination sphere of Mn catalase, CW Q-band ENDOR spectroscopy revealed two distinctly different (17)O signals incorporated in distinctly different time regimes. First, a signal appearing after 2 h of (17)O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the (17)O µ-oxo hyperfine coupling of the di-µ-oxo di-Mn(III,IV) bipyridine model (Usov, O. M.; Grigoryants, V. M.; Tagore, R.; Brudvig, G. W.; Scholes, C. P. J. Am. Chem. Soc. 2007, 129, 11886-11887), this signal was assigned to µ-oxo oxygen. EPR line broadening was obvious from this (17)O µ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-µ-oxo position, from which (17)O ENDOR showed a smaller 3.8 MHz hyperfine coupling and possible quadrupole splittings, indicating a terminal water of Mn(III). Exchangeable proton/deuteron hyperfine couplings, consistent with terminal water ligation to Mn(III), also appeared. Q-band CW ENDOR from the S(2) state of the OEC was obtained following multihour (17)O exchange, which showed a (17)O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as µ-oxo-(17)O by resemblance to the µ-oxo signals from Mn catalase and the di-µ-oxo di-Mn(III,IV) bipyridine model.
Assuntos
Catalase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Lactobacillus plantarum/enzimologia , Complexo de Proteína do Fotossistema II/metabolismo , Água/metabolismo , Catalase/química , Lactobacillus plantarum/química , Modelos Moleculares , Oxirredução , Complexo de Proteína do Fotossistema II/química , Água/químicaRESUMO
Non-heme manganese catalases are widely distributed over microbial life and represent an environmentally important alternative to heme-containing catalases in antioxidant defense. Manganese catalases contain a binuclear manganese complex as their catalytic active site rather than a heme, and cycle between Mn(2)(II,II) and Mn(2)(III,III) states during turnover. X-ray crystallography has revealed the key structural elements of the binuclear manganese active site complex that can serve as the starting point for computational studies on the protein. Four manganese catalase enzymes have been isolated and characterized, and the enzyme appears to have a broad phylogenetic distribution including both bacteria and archae. More than 100 manganese catalase genes have been annotated in genomic databases, although the assignment of many of these putative manganese catalases needs to be experimentally verified. Iron limitation, exposure to low levels of peroxide stress, thermostability and cyanide resistance may provide the biological and environmental context for the occurrence of manganese catalases.
Assuntos
Catalase/química , Heme/química , Manganês/química , Antioxidantes/química , Bioquímica/métodos , Domínio Catalítico , Cristalografia por Raios X/métodos , Lactobacillus plantarum/enzimologia , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Estresse Oxidativo , Filogenia , Espécies Reativas de Oxigênio , Solventes/química , Termodinâmica , Thermus/enzimologiaRESUMO
Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn2)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide-crosslink, exhibits anti-cooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (K(D)=1×10â»5 M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro.
Assuntos
Escherichia coli/enzimologia , Manganês/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Transporte Biológico , Cromatografia em Gel , Cristalografia por Raios X , Dissulfetos/química , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Pulse electron paramagnetic resonance (EPR) spectroscopy is employed at two very different excitation frequencies, 9.77 and 30.67 GHz, in the study of the nitrogen coordination environment of the Mn(III)Mn(IV) state of the dimanganese-containing catalases from Lactobacillus plantarum and Thermus thermophilus. Consistent with previous studies, the lower-frequency results reveal one unique histidine nitrogen-Mn cluster interaction. For the first time, a second, more strongly hyperfine-coupled (14)N atom is unambiguously observed through the use of higher frequency/higher field EPR spectroscopy. The low excitation frequency spectral features are rationalized as arising from the interaction of a histidine nitrogen that is bound to the Mn(IV) ion, and the higher excitation frequency features are attributed to the histidine nitrogen bound to the Mn(III) ion. These results allow for the computation of intrinsic hyperfine coupling constants, which range from 2.2 to 2.9 MHz, for sp(2)-hybridized nitrogens coordinating equatorially to high-valence Mn ions. The relevance of these findings is discussed in the context of recent results from analogous higher frequency EPR studies of the Mn cluster in photosystem II and other exchange-coupled, transition metal-containing systems.
Assuntos
Catalase/química , Catalase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Nitrogênio/química , Domínio Catalítico , Lactobacillus plantarum/enzimologia , Manganês/metabolismo , Modelos Moleculares , Thermus thermophilus/enzimologiaRESUMO
To determine the radiation sensitivity of galactose oxidase, a 68 kDa monomeric enzyme containing a mononuclear copper ion coordinated with an unusually stable cysteinyl-tyrosine (Cys-Tyr) protein free radical. Both active enzyme and reversibly rendered inactive enzyme were irradiated in the frozen state with high-energy electrons. Surviving polypeptides and surviving enzyme activity were analyzed by radiation target theory giving the radiation sensitive mass for each property. In both active and inactive forms, protein monomer integrity was lost with a single radiation interaction anywhere in the polypeptide, but enzymatic activity was more resistant, yielding target sizes considerably smaller than that of the monomer. These results suggest that the structure of galactose oxidase must make its catalytic activity unusually robust, permitting the enzymatic properties to survive in molecules following cleavage of the polymer chain. Radiation target size for loss of monomers yielded the mass of monomers indicating a polypeptide chain cleavage after a radiation interaction anywhere in the monomer. Loss of enzymatic activity yielded a much smaller mass indicating a robust structure in which catalytic activity could be expressed in cleaved polypeptides.
Assuntos
Galactose Oxidase/metabolismo , Ascomicetos/enzimologia , Biocatálise , Ativação Enzimática/efeitos da radiação , Radicais Livres/metabolismo , Peso MolecularRESUMO
Manganese superoxide dismutase is an important antioxidant defense metalloenzyme that protects cells from damage by the toxic oxygen metabolite, superoxide free radical, formed as an unavoidable by-product of aerobic metabolism. Many years of research have gone into understanding how the metal cofactor interacts with small molecules in its catalytic role. In contrast, very little is presently known about how the protein acquires its metal cofactor, an important step in the maturation of the protein and one that is absolutely required for its biological function. Recent work is beginning to provide insight into the mechanisms of metal delivery to manganese superoxide dismutase in vivo and in vitro.
Assuntos
Metais/metabolismo , Superóxido Dismutase/metabolismo , Animais , Transporte Biológico , HumanosRESUMO
Metal uptake by the antioxidant defense metalloenzyme manganese superoxide dismutase (MnSOD) is an essential step in the functional maturation of the protein that is just beginning to be investigated in detail. We have extended earlier in vitro studies on metal binding by the dimeric Escherichia coli apo-MnSOD to investigate the mechanism of metal uptake by tetrameric human and Thermus thermophilus apo-MnSODs. Like the E. coli apo-MnSOD, these proteins also bind metal ions in vitro in a thermally activated, pH-sensitive process. However, metal uptake by the tetrameric apo-MnSODs exhibits a number of important differences. In particular, there is no indication of conformational gating requirement for metal binding for these proteins, and the reaction is first-order in metal ion. The high concentration of metal ion that is required to achieve physiologically relevant metallation rates for tetrameric human apo-MnSOD in vitro suggests the possibility that co-translational metal binding or chaperone interactions may be required in vivo.
Assuntos
Metais/metabolismo , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Superóxido Dismutase/química , Temperatura , Thermus thermophilus/enzimologiaRESUMO
Metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. This work systematically explores the structural determinants of metal binding in both wild-type (WT) apoprotein and mutational variants as a test of mechanistic models. The pH dependence of metalation under physiological conditions (37 degrees C) shows it is linked to ionization of a single proton with a p K a of 7.7. Size exclusion chromatography demonstrates that the apoprotein is dimeric even when it is fully converted to the open form. The role of molecular motions in metal binding has been probed by using disulfide engineering to introduce covalent constraints into the protein. While restricting motion at domain interfaces has no effect, constraining the subunit interface significantly perturbs metal uptake but does not prevent the process. Mutagenesis of residues in the active site environment results in a dramatic shift in the transition temperature by as much as 20 degrees C or a loss of pH sensitivity. On the basis of these results, a mechanism for metal uptake by manganese superoxide dismutase involving reorientation of active site residues to form a metal entry channel is proposed.
Assuntos
Superóxido Dismutase/química , Substituição de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Domínio Catalítico/genética , Dimerização , Concentração de Íons de Hidrogênio , Transporte de Íons , Cinética , Metais/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , TermodinâmicaRESUMO
Galactose oxidase is a metalloenzyme containing a novel metalloradical complex in its active site, comprised of a mononuclear copper ion associated with a protein free radical. The free radical has been shown to be localized on an intrinsic redox cofactor, 3'-(S-cysteinyl)tyrosine (Cys-Tyr), formed by a posttranslational covalent coupling of tyrosine and cysteine side chains in a self-processing reaction. The role of the thioether linkage in the function of the Cys-Tyr cofactor is unresolved, and some computational studies have suggested that the thioether substituent has a negligible effect on the properties of the tyrosyl free radical. In order to address this question experimentally, we have incorporated site-selectively labeled tyrosine ((2)H, (13)C, (17)O) into galactose oxidase using an engineered tyrosine auxotroph strain of Pichia pastoris . (33)S was also incorporated into the protein. EPR spectra for the Cys-Tyr(*) free radical in each of these isotopic variants were analyzed to extract nuclear hyperfine parameters for comparison with theoretical predictions, and the unpaired spin distribution in the free radical was reconstructed from the hyperfine data. These labeling studies allow the first comprehensive experimental evaluation of the effect of the thioether linkage on the properties of Cys-Tyr(*) and indicate that previous calculations significantly underestimated the contribution of this feature to the electronic ground state of the free radical.
Assuntos
Dipeptídeos/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Radicais Livres/química , Galactose Oxidase/química , Algoritmos , Isótopos de Carbono/química , Catálise , Domínio Catalítico , Cobre/química , Deutério/química , Galactose Oxidase/genética , Galactose Oxidase/metabolismo , Cinética , Metaloproteínas/química , Metaloproteínas/metabolismo , Modelos Químicos , Modelos Moleculares , Oxirredução , Isótopos de Oxigênio/química , Pichia/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Isótopos de Enxofre/químicaRESUMO
Labeling proteins with stable isotopes is important for many analytical and structural techniques, including NMR spectroscopy and mass spectrometry. Nonselective labeling, which uniformly labels all amino acids in the protein, may be accomplished with readily available wild-type expression hosts. However, there are often advantages to labeling a specific amino acid, and residue-selective labeling generally requires the use of an expression strain that is auxotrophic for the amino acid in order to efficiently incorporate the isotopic label. The behavior of an auxotrophic strain may be complicated by the regulatory properties of the biosynthetic pathway, by secondary nutritional requirements resulting from disruption of a biosynthetic pathway, and from acquired sensitivity to environmental factors resulting from build-up of metabolic intermediates. As a result, it is important to characterize the phenotype of the each auxotrophic strain in order to optimize its performance as an expression host for selective labeling of proteins. The application of aromatic auxotroph strains of Pichia pastoris to labeling tyrosines in a recombinant protein (galactose oxidase) will be used to illustrate selective-labeling methods.
Assuntos
Aminoácidos/metabolismo , Processos Autotróficos , Marcação por Isótopo/métodos , Proteínas Recombinantes/metabolismo , Aminoácidos/biossíntese , Espectroscopia de Ressonância de Spin Eletrônica , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Transformação GenéticaRESUMO
High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.
Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Pichia/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Engenharia de Proteínas/métodos , Triticum/enzimologia , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Triticum/genéticaRESUMO
Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O(2)-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.
Assuntos
Manganês/química , Oxirredutases/metabolismo , Sítios de Ligação , Catálise , Dicroísmo Circular , Óxido de Deutério/química , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Oxirredutases/químicaRESUMO
Chitosan was chosen as a natural polymer for dispersion of single walled carbon nanotubes (SWNT) based on its ability to efficiently solubilize SWNTs to form a stable dispersion. Moreover, chitosan films deposited on a surface of a glassy carbon (GC) electrode are mechanically stable. Further stabilisation of the chitosan film containing SWNT (CHIT-SWNT) was done by chemical crosslinking with glutaraldehyde and free aldehyde groups produced a substrate used for covalent immobilisation of galactose oxidase (GalOD). Different galactose biosensor configurations were tested with optimisation of composition of inner and outer membrane; and enzyme immobilisation procedure, as well. Detection of oxygen uptake by GalOD on CHIT-SWNT layer at -400 mV is robust and, when flow injection analysis (FIA) was applied for assays, a low detection limit (25 microM) and very high assay throughput rate (150 h-1) was achieved. This new galactose biosensor offers highly reliable detection of galactose with R.S.D. well below 2% and it has been successfully applied to assaying galactose in a blood sample with recovery index between 101.2 and 102.7%.
Assuntos
Técnicas Biossensoriais/métodos , Quitosana , Galactose/análise , Nanotubos de Carbono , CalibragemRESUMO
The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.