Characterization of Euphorbia characias Latex Amine Oxidase1

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105 m−1 cm−1 and 6000 m−1 cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

Molecular Characterization of the Oxalate Oxidase Involved in the Response of Barley to the Powdery Mildew Fungus1

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein–protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an α-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or α-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen: the role of a bound ubiquinone molecule.

We have studied the kinetics of the oxygen reaction of the fully reduced quinol oxidase, cytochrome bo3, using flow-flash and stopped flow techniques. This enzyme belongs to the heme-copper oxidase family but lacks the CuA center of the cytochrome c oxidases. Depending on the isolation procedure, the kinetics are found to be either nearly monophasic and very different from those of cytochrome c oxidase or multiphasic and quite similar to cytochrome c oxidase. The multiphasic kinetics in cytochrome c oxidase can largely be attributed to the presence Of CuA as the donor of a fourth electron, which rereduces the originally oxidized low-spin heme and completes the reduction of O2 to water. Monophasic kinetics would thus be expected, a priori, for cytochrome bo3 since it lacks the CuA center, and in this case we show that the oxygen reaction is incomplete and ends with the ferryl intermediate. Multiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CuA). We observe such kinetics exclusively with cytochrome bo3 that contains a single equivalent of bound ubiquinone-8, whereas we find no bound ubiquinone in an enzyme exhibiting monophasic kinetics. Reconstitution with ubiquinone-8 converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiquinone molecule in cytochrome bo3 is capable of fast rereduction of heme b and that the reaction with O2 is quite similar in quinol and cytochrome c oxidases.

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.

Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site.

Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region. The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.

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 GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Espécies polinucleares de cobre e ferro com catalisadores de oxidação e modelos de sítios ativos

Diferentes complexos de cobre(II), contendo ligantes do tipo base de Schiff e um grupamento imidazólico, com interesse bioinorgânico, catalítico e como novos materiais, foram preparados na forma de sais perclorato, nitrato ou cloreto e caracterizados através de diferentes técnicas espectroscópicas (UV/Vis, IR, EPR, Raman) e espectrometria de massa Tandem (ESI-MS/MS), além de análise elementar, condutividade molar e medidas de propriedades magnéticas. Alguns destes compostos, obtidos como cristais adequados, tiveram suas estruturas determinadas por cristalografia de raios-X. As espécies di- e polinucleares contendo pontes cloreto, mostraram desdobramentos das hiperfinas nos espectros de EPR, relacionados à presença do equilíbrio com a respectiva espécie mononuclear, devido à labilidade dos íons cloretos, dependendo do contra-íon e do tipo de solvente utilizado. Adicionalmente, em solução alcalina, estes compostos estão em equilíbrio com as correspondentes espécies polinucleares, onde os centros de cobre estão ligados através de um ligante imidazolato. Em meio alcalino, estes compostos polinucleares contendo ponte imidazolato foram também isolados e caracterizados por diferentes técnicas espectroscópicas e magnéticas. Através da variação estrutural e também do ligante-ponte foi possível modular o fenômeno da interação magnética entre os íons de cobre em estruturas correlatas di- e polinucleares. Os respectivos parâmetros magnéticos foram obtidos com ajuste das curvas experimentais de XM vs T, correlacionando-se muito bem com a geometria, ângulos e distâncias de ligação entre os íons, quando comparado com outros complexos similares descritos na literatura. Posteriormente, estudaram-se os fatores relacionados com a reatividade de todas essas espécies como catalisadores na oxidação de substratos de interesse (fenóis e aminas), através da variação do tamanho da cavidade nas estruturas cíclicas ou de variações no ligante coordenado ao redor do íon metálico. Vários deles se mostraram bons miméticos de tirosinases e catecol oxidases. Um novo complexo-modelo da citocromo c oxidase (CcO), utilizando a protoporfirina IX condensada ao quelato N,N,-bis[2-(1,2-metilbenzimidazolil)etil]amino e ao resíduo de glicil-L-histidina, foi sintetizado e caracterizado através de diferentes técnicas espectroscópicas, especialmente EPR. A adição de H2O2 ao sistema completamente oxidado, FeIII/CuII, a -55°C, ou o borbulhamento de oxigênio molecular a uma solução do complexo na sua forma reduzida, FeII/CuI, saturada de CO, resultou na formação de adutos com O2, de baixo spin, estáveis a baixas temperaturas.

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hydrogen peroxide is a substrate or side-product in many enzyme-catalyzed reactions. For example, it is a side-product of oxidases, resulting from the re-oxidation of FAD with molecular oxygen, and it is a substrate for peroxidases and other enzymes. However, hydrogen peroxide is able to chemically modify the peptide core of the enzymes it interacts with, and also to produce the oxidation of some cofactors and prostetic groups (e.g., the hemo group). Thus, the development of strategies that may permit to increase the stability of enzymes in the presence of this deleterious reagent is an interesting target. This enhancement in enzyme stability has been attempted following almost all available strategies: site-directed mutagenesis (eliminating the most reactive moieties), medium engineering (using stabilizers), immobilization and chemical modification (trying to generate hydrophobic environments surrounding the enzyme, to confer higher rigidity to the protein or to generate oxidation-resistant groups), or the use of systems capable of decomposing hydrogen peroxide under very mild conditions. If hydrogen peroxide is just a side-product, its immediate removal has been reported to be the best solution. In some cases, when hydrogen peroxide is the substrate and its decomposition is not a sensible solution, researchers coupled one enzyme generating hydrogen peroxide “in situ” to the target enzyme resulting in a continuous supply of this reagent at low concentrations thus preventing enzyme inactivation. This review will focus on the general role of hydrogen peroxide in biocatalysis, the main mechanisms of enzyme inactivation produced by this reactive and the different strategies used to prevent enzyme inactivation caused by this “dangerous liaison”.

Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization

Metallosphaera sedula is a thermoacidophilic Crenarchaeon which is capable of leaching metals from sulfidic ores. The authors have investigated the presence and expression of genes encoding respiratory complexes in this organism when grown heterotrophically or chemolithotrophically on either sulfur or pyrite. The presence of three gene clusters, encoding two terminal oxidase complexes, the quinol oxidase SoxABCD and the SoxM oxidase supercomplex, and a gene cluster encoding a high-potential cytochrome b and components of a bc(1) complex analogue (cbsBA-soxL2N gene cluster) was established. Expression studies showed that the soxM gene was expressed to high levels during heterotrophic growth of M. sedula on yeast extract, while the soxABCD mRNA was most abundant in cells grown on sulfur. Reduced-minus-oxidized difference spectra of cell membranes showed cytochrome-related peaks that correspond to published spectra of Sulfolobus-type terminal oxidase complexes. In pyrite-grown cells, expression levels of the two monitored oxidase gene clusters were reduced by a factor of 10-12 relative to maximal expression levels, although spectra of membranes clearly contained oxidase-associated haems, suggesting the presence of additional gene clusters encoding terminal oxidases in M. sedula. Pyrite- and sulfur-grown cells contained high levels of the cbsA transcript, which encodes a membrane-bound cytochrome b with a possible role in iron oxidation or chemolithotrophy. The cbsA gene is not co-transcribed with the soxL2N genes, and therefore does not appear to be an integral part of this bc(1) complex analogue. The data show for the first time the differential expression of the Sulfolobus-type terminal oxidase gene clusters in a Crenarchaeon in response to changing growth modes.

Pulsed EPR studies of a bacterial sulfite-oxidizing enzyme with pH-invariant hyperfine interactions from exchangeable protons

Variable-frequency pulsed electron paramagnetic resonance studies of the molybdenum(V) center of sulfite dehydrogenase (SDH) clearly show couplings from nearby exchangeable protons that are assigned to a (MoOHn)-O-v group. The hyperfine parameters for these exchangeable protons of SDH are the same at both low and high pH and similar to those for the high-pH forms of sulfite oxidases (SOs) from eukaryotes. The SDH proton parameters are distinctly different from the low-pH forms of chicken and human so.

Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulfite dehydrogenase

The sulfite dehydrogenase from Starkeya novella is the only known sulfite-oxidizing enzyme that forms a permanent heterodimeric complex between a molybdenum and a heme c-containing subunit and can be crystallized in an electron transfer competent conformation. Tyr236 is a highly conserved active site residue in sulfite oxidoreductases and has been shown to interact with a nearby arginine and a molybdenum-oxo ligand that is involved in catalysis. We have created a Tyr236 to Phe substitution in the SorAB sulfite dehydrogenase. The purified SDHY236F protein has been characterized in terms of activity, structure, intramolecular electron transfer, and EPR properties. The substituted protein exhibited reduced turnover rates and substrate affinity as well as an altered reactivity toward molecular oxygen as an electron acceptor. Following reduction by sulfite and unlike SDHWT, the substituted enzyme was reoxidized quickly in the presence of molecular oxygen, a process reminiscent of the reactions of the sulfite oxidases. SDHY236F also exhibited the pH-dependent CW-EPR signals that are typically observed in vertebrate sulfite oxidases, allowing a direct link of CW-EPR properties to changes caused by a single-amino acid substitution. No quantifiable electron transfer was seen in laser flash photolysis experiments with SDHY236F. The crystal structure of SDHY236F clearly shows that as a result of the substitution the hydrogen bonding network surrounding the active site is disturbed, resulting in an increased mobility of the nearby arginine. These disruptions underline the importance of Tyr236 for the integrity of the substrate binding site and the optimal alignment of Arg55, which appears to be necessary for efficient electron transfer.

Structure of the active site of sulfite dehydrogenase from Starkeya novella

In this paper, we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella. Our results provide the first direct structural information on the active site of the oxidized form of this enzyme and confirm the conclusions derived from protein crystallography that the molybdenum coordination is analogous to that of the sulfite oxidases. The molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 angstrom and three thiolate Mo-S ligands at 2.42 angstrom, whereas the reduced enzyme has one oxo at 1.74 angstrom, one long oxygen at 2.19 angstrom (characteristic of Mo-OH2), and three Mo-S ligands at 2.40 angstrom.