Purification and Characterization of a Prokaryotic Xanthine Dehydrogenase from Comamonas acidovorans

Mammalian molybdo-flavoenzymes are oxidases requiring FAD and molybdopterin (molybdenum cofactor) for their catalytic activity. This family of proteins was thought to consist of four members, xanthine oxidoreductase, aldehyde oxidase 1 (AOX1), and the aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2, respectively). Whereas the first two enzymes are present in humans and various other mammalian species, the last two proteins have been described only in mice. Here, we report on the identification, in both mice and rats, of a novel molybdo-flavoenzyme, AOH3. In addition, we have cloned the cDNAs coding for rat AOH1 and AOH2, demonstrating that this animal species has the same complement of molybdo-flavoproteins as the mouse. The AOH3 cDNA is characterized by remarkable similarity to AOX1, AOH1, AOH2, and xanthine oxidoreductase cDNAs. Mouse AOH3 is selectively expressed in Bowman's glands of the olfactory mucosa, although small amounts of the corresponding mRNA are present also in the skin. In the former location, two alternatively spliced forms of the AOH3 transcript with different 3-untranslated regions were identified. The general properties of AOH3 were determined by purification of mouse AOH3 from the olfactory mucosa. The enzyme possesses aldehyde oxidase activity and oxidizes, albeit with low efficiency, exogenous substrates that are recognized by AOH1 and AOX1. The Aoh3 gene maps to mouse chromosome 1 band c1 and rat chromosome 7 in close proximity to the Aox1, Aoh1, and Aoh2 loci and has an exon/intron structure almost identical to that of the other molybdo-flavoenzyme genes in the two species.

Section: Aoh3 a Novel Molybdo-flavoenzyme 50495supporting

confidence: 85%

The Aldehyde Oxidase Gene Cluster in Mice and Rats

Kurosaki

Terao

Barzago

et al. 2004

“…Steady 16) and is similar to that of Comamonas acidovorans XDH (18). The bacterial enzymes thus react much faster with xanthine as substrate, which explains the higher activities (expressed as AFR) of the enzyme in standard assays as compared with bovine milk XO that have been described previously (6).…”

Section: Purification Of Active R Capsulatus Xdh By Folate Affinitysupporting

confidence: 60%

The Role of Active Site Glutamate Residues in Catalysis of Rhodobacter capsulatus Xanthine Dehydrogenase

Leimkühler

Stockert

Igarashi

et al. 2004

104

Xanthine dehydrogenase (XDH) from the bacteriumRhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD ؉ as the electron acceptor. R. capsulatus XDH forms an (␣␤) 2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. Xanthine dehydrogenase (XDH, 1 EC 1.1.1.204) catalyzes the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid, the last two steps in the formation of urate. A water molecule rather than O 2 is the ultimate source of oxygen incorporated into the product, and the reaction is thus fundamentally different from that of all other monooxygenase/hydroxylase systems. The mammalian enzyme is synthesized as the dehydrogenase form, XDH, and exists mostly as such in the cell, although it can readily be converted to an oxidase form (XO) by oxidation of sulfhydryl residues or by proteolysis (1, 2). XDH shows a preference for NAD ϩ reduction at the FAD site but is also able to react with molecular oxygen, whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate. The conversion of XDH to XO is of considerable medical interest, because the enzyme is implicated in hyperuricemia, and its activity is proposed to play a role in postischemic reperfusion injury (3). The mammalian enzyme is a homodimer with a molecular mass of 290 kDa, with each monomer acting independently in catalysis. Each subunit contains one molybdenum cofactor (Moco, consisting of a MoOS(OH) core coordinated to 1 eq of molybdopterin), two [2Fe-2S] clusters that are distinguishable by EPR, and 1 eq of FAD (4). The oxidation of xanthine takes place at the Moco, and the electrons thus introduced are transferred via the two FeS centers to FAD by intramolecular electron transfer.Rhodobacter capsulatus XDH is a cytoplasmic enzyme with an (␣␤) 2 heterodimeric structure, with the two subunits encoded by the xdhA and xdhB genes, respectively (5). The redox-active cofactors are bound to two different subunits; the FAD and [2Fe2S] centers are found in the XDHA subunits, and the Moco is found in the XDHB subunits. This complement of redox-active centers provides the enzyme with the capacity to hold a maximum of 6 reducing eq per functioning ␣␤ unit (corresponding to one intact subunit in the case of the vertebrate enzymes) upon reduction; under anaerobic conditions each can react with 3 eq of xanthine. Unlike the mammalian oxidoreductases, but like avian XDH, R. capsulatus XDH is isolated with high reactivity toward NAD ϩ and does not undergo the conversion to the oxidase form (6). In addition to xanthine, XDH is reducible by NADH (7, 8).The recently solved crystal structures of the mammalian xanthine oxidoreductase (XOR) from bovine milk (9) and the homologous bacterial XDH from R. capsulatus (10) have pav...

“…On the reduction with dithionite, the spectrum changes markedly with less intense transitions at 371 (Ϫ), 409 (ϩ), 461 (Ϫ), and 573 (Ϫ) nm. The visible CD spectra of reduced and oxidized R. capsulatus wild-type XDH are very similar in shape and intensity to those of Comamonas acidovorans XDH (26). The spectra of the two variants were measured and compared with wildtype XDH.…”

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

confidence: 99%

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

Schumann

Saggu

Möller

et al. 2008

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...