Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation–reduction potentials of its redox-active centers, and first EPR characterization

Parschat, Katja; Canne, Christoph; Hüttermann, Jürgen; Kappl, Reinhard; Fetzner, Susanne

doi:10.1016/s0167-4838(00)00214-4

Cited by 33 publications

(28 citation statements)

References 66 publications

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“…The spectra display signals from several superimposed paramagnetic species, which are observed usually in enzymes belonging to the XO family. However, the most prominent signals are the characteristic EPR signals assigned to the two iron-sulfur centers FeSI and FeSII, which are similar for all of the members of the XO family that have been described to date (Hille, 1996;Parschat et al, 2001). FeSI has EPR properties showing a slightly rhombic g tensor, similar to those of many other [2Fe2S] proteins, being fully developed at relatively high temperatures (up to T ϭ 80 K), whereas FeSII has unusual EPR properties for [2Fe2S] species with a more rhombic g tensor, showing lines with varying line widths that can only be observed clearly at much lower temperatures (T Ͻ 30 K).…”

Section: Epr Spectroscopy Of the Haox1 [2fe2s]mentioning

confidence: 83%

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

et al. 2012

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ABSTRACT:Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, was obtained with a purity of 95% and a yield of 50 g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.

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Section: Epr Spectroscopy Of the Haox1 [2fe2s]mentioning

confidence: 83%

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

et al. 2012

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“…This line width is larger as compared with that from usual flavins (ϳ1.9 mT) (11) and points to a magnetic interaction observed at low temperatures with the metal centers. At 80 K the line width of FAD is only 2.1 mT, in accordance to that from other FAD cofactors (11). The Moco (Mo V ) has been neglected in the simulations.…”

Section: Investigations Of the Fes Clusters Of (␣␤) Heterodimeric Andmentioning

confidence: 58%

The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsulatus Xanthine Dehydrogenase

Schumann

Saggu

Möller

et al. 2008

Journal of Biological Chemistry

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Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (␣␤) 2 heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (␣␤) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (␣␤) subunits has to precede Moco insertion. In an (␣␤) 2 XDH heterotetramer variant, including only one active Moco-center, the active (␣␤) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (␣␤) 2 heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (␣␤) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation. Xanthine oxidoreductases (XORs)2 are the best studied enzymes of the small but important class of molybdenum-containing iron-sulfur flavoproteins, containing two non-identical [2Fe2S] clusters, FAD, and the sulfurated form of the molybdenum cofactor (Moco) as catalytically acting units (1-3).Mammalian XORs catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, and exist originally as the dehydrogenase form (XDH, EC 1.17.1.4) but can be converted to the oxidase form (XO, EC 1.1.3.22) either reversibly by oxidation of sulfhydryl residues of the protein molecule or irreversibly by proteolysis (4). XDH shows a preference for NAD ϩ reduction at the FAD reaction site, whereas XO exclusively uses dioxygen as a terminal electron acceptor, leading to the formation of superoxide and hydrogen peroxide (5). The enzyme has been implicated in diseases characterized by oxygen radical-induced tissue damage, such as postischemic reperfusion injury (6). The oxidation of xanthine takes place at the molybdenum center, and the electrons thus introduced are rapidly distributed to the other centers according to their relative redox potentials (1). The re-oxidation of the reduced enzyme by the oxidant substrate, either NAD ϩ or molecular oxygen, occurs through FAD (7). The two [2Fe2S] clusters (FeSI and FeSII) are indistinguishable in terms of their absorption spectra, but the midpoint redox potential of FeSII is generally more positive than that of the FeSI center (8, 9). The FeS centers from enzymes of the XO family have been characterized earlier by EPR (10 -12). The FeSI center of eukaryotic XOR exh...

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“…The spectra display signals from several superimposed paramagnetic species, which are usually observed for enzymes of the XO family. However, the most prominent signals are the characteristic EPR signals assigned to the two iron sulfur centers FeSI and FeSII, which are similar for all of the members of the XO family that have been described to date (Hille, 1996;Parschat et al, 2001). FeSI has EPR properties showing a rhombic g tensor similar to those of many other [2Fe-2S] proteins, being fully developed at relatively high temperatures (up to T ϭ 80 K), whereas FeSII has unusual EPR properties for [2Fe-2S] species with a strongly rhombic g tensor, showing broad lines that can only be observed clearly at much lower temperatures (T Ͻ 40 K).…”

Section: Purification Of Recombinant Maox3 After Expression In E Colmentioning

confidence: 78%

Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver toEscherichia coliHeterologous Protein Expression

et al. 2011

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ABSTRACT:Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N 1 -methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 Å. The crystals belong to space group P1, and two dimers are present in the unit cell.

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Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation–reduction potentials of its redox-active centers, and first EPR characterization

Cited by 33 publications

References 66 publications

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsulatus Xanthine Dehydrogenase

Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver toEscherichia coliHeterologous Protein Expression

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