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Query: UNIPROT:P06889 (Mol)
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Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe-2S] clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding sigma54) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N-terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an alpha2beta2-subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.
Mol Microbiol 1998 Feb
PMID:Xanthine dehydrogenase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes. 951 10

We describe the sequence changes of a number of mutations of the Aspergillus nidulans xanthine dehydrogenase (XDH). We have located the amino acids affected by these changes in the three-dimensional (3D) structure of aldehyde oxido-reductase (MOP) from Desulfovibrio gigas, related to eukaryotic XDHs. Of these, two are loss of function mutations, mapping, respectively, in the molybdenum-pterin co-factor (MoCo) domain and in the domain involved in substrate recognition. Changes in two amino acids result in resistance to the irreversible inhibitor allopurinol. In Arg911 two different changes, conserved among all XDHs and MOP but not in other aldehyde oxidases (AO), change the position of hydroxylation of the analogue 2-hydroxypurine from C-8 to C-6. A number of changes affect residues adjacent to the molybdenum or its ligands. Arg911 is positioned in the substrate pocket in a way that it can account for the positioning of purine substrates in relation to the MoCo reactive center, together with a glutamate residue, universally conserved among the XDHs (Glu833).
J Mol Biol 1998 May 01
PMID:Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase. 957 Oct 62

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.
Mol Microbiol 1998 May
PMID:NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. 963 49

The nitrogenase iron (Fe) protein performs multiple functions during biological nitrogen fixation, including mediating the mechanistically essential coupling between ATP hydrolysis and electron transfer to the nitrogenase molybdenum iron (MoFe) protein during substrate reduction, and participating in the biosynthesis and insertion of the FeMo-cofactor into the MoFe-protein. To establish a structural framework for addressing the diverse functions of Fe-protein, crystal structures of the Fe-proteins from Azotobacter vinelandii and Clostridium pasteurianum have been determined at resolutions of 2.2 A and 1.93 A, respectively. These two Fe-proteins are among the more diverse in terms of amino acid sequence and biochemical properties. As described initially for the A. vinelandii Fe-protein in a different crystal form at 2.9 A resolution, each subunit of the dimeric Fe-protein adopts a polypeptide fold related to other mononucleotide-binding proteins such as G-proteins, with the two subunits bridged by a 4Fe:4S cluster. The overall similarities in the subunit fold and dimer arrangement observed in the structures of the A. vinelandii and C. pasteurianum Fe-proteins indicate that they are representative of the conformation of free Fe-protein that is not in complex with nucleotide or the MoFe-protein. Residues in the cluster and nucleotide-binding sites are linked by a network of conserved hydrogen bonds, salt-bridges and water molecules that may conformationally couple these regions. Significant variability is observed in localized regions, especially near the 4Fe:4S cluster and the MoFe-protein binding surface, that change conformation upon formation of the ADP.AlF4- stabilized complex with the MoFe-protein. A core of 140 conserved residues is identified in an alignment of 59 Fe-protein sequences that may be useful for the identification of homologous proteins with functions comparable to that of Fe-protein in non-nitrogen fixing systems.
J Mol Biol 1998 Jul 24
PMID:Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum. 967 96

Physiological and genetic characterization of Staphylococcus carnosus nitrate reductase-negative mutants led to the identification of the nitrate reductase operon, narGHJI. Transcription from the nar promoter was stimulated by anaerobiosis, nitrate, and nitrite. This is in accordance with the nitrate reductase activities determined with benzyl viologen as electron donor. However, in the presence of oxygen and nitrate, high transcriptional initiation but low nitrate reductase activity was observed. Since the alphabeta complex of the nitrate reductase formed during anaerobic growth was insensitive to oxygen, other oxygen-sensitive steps (e.g., post-transcriptional mechanisms, molybdenum cofactor biosynthesis) must be involved. The nitrate-reducing system in S. carnosus displays similarities to the dissimilatory nitrate reductases of Escherichia coli. However, in the S. carnosus nar promoter, no obvious Fnr and integration host factor recognition sites are present; only one site that is related to the E. coli NarL consensus sequence was found. Studies to determine whether the E. coli proteins NarL and Fnr are functional at the S. carnosus narGHJI promoter indicated that the promoter is not functional in E. coli.
Mol Gen Genet 1998 Jul
PMID:Identification and characterization of the Staphylococcus carnosus nitrate reductase operon. 973 86

Trimethylamine N-oxide (TMAO) is an abundant compound of tissues of marine fish and invertebrates. During fish spoilage, certain marine bacteria can reduce TMAO to nauseous trimethylamine (TMA). One such bacterium has been isolated and identified as a new Shewanella species, and called Shewanella massilia. The anaerobic growth of S. massilia is greatly increased when TMAO is added, indicating that TMAO reduction involves a respiratory pathway. The TorA enzyme responsible for TMAO reduction is a molybdenum cofactor-containing protein of 90 kDa located in the periplasm. Whereas TorA is induced by both TMAO and dimethylsulfoxide (DMSO), this enzyme has a high substrate specificity and appears to only efficiently reduce TMAO as a natural compound. The structural torA gene encoding the TMAO reductase (TorA) and its flanking regions were amplified using PCR techniques. The torA gene is the third gene of a TMAO-inducible operon (torECAD) encoding the TMAO respiratory components. The torC gene, located upstream from torA encodes a pentahemic c-type cytochrome, likely to be involved in electron transfer to the TorA terminal reductase. TorC was shown to be anchored to the membrane and, like TorA, is induced by TMAO. Except for the TorE protein, which is encoded by the first gene of the torECAD operon, all the tor gene products are homologous to proteins found in the TMAO/DMSO reductase systems from Escherichia coli and Rhodobacter species. In addition, the genetic organization of these systems is similar. Although these bacteria are found in different ecological niches, their respiratory systems appear to be phylogenetically related, suggesting that they come from a common ancestor.
J Mol Biol 1998 Nov 27
PMID:Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory system from a Shewanella species. 981 27

The periplasmic trimethylamine N-oxide (TMAO) reductase from the marine bacteria Shewanella massilia is involved in a respiratory chain, having trimethylamine N-oxide as terminal electron acceptor. This molybdoenzyme belongs to the dimethyl sulfoxide (DMSO) reductase family, but has a different substrate specificity than its homologous enzyme. While the DMSO reductases reduce a broad spectra of organic S-oxide and N-oxide compounds, TMAO reductase from Shewanella massilia reduces only TMAO as the natural compound. The crystal structure was solved by molecular replacement with the coordinates of the DMSO reductase from Rhodobacter sphaeroides. The overall fold of the protein structure is essentially the same as the DMSO reductase structures, organized into four domains. The molybdenum coordination sphere is closest to that described in the DMSO reductase of Rhodobacter capsulatus. The structural differences found in the protein environment of the active site could be related to the differences in substrate specificity of these enzymes. In close vicinity of the molybdenum ion a tyrosine residue is missing in the TMAO reductase, leaving a greater space accessible to the solvent. This tyrosine residue has contacts to the oxo groups in the DMSO reductase structures. The arrangement and number of charged residues lining the inner surface of the funnel-like entrance to the active site, is different in the TMAO reductase than in the DMSO reductases from Rhodobacter species. Furthermore a surface loop at the top of the active-site funnel, for which no density was present in the DMSO reductase structures, is well defined in the oxidized form of the TMAO reductase structure, and is located on the border of the funnel-like entrance of the active center.
J Mol Biol 1998 Nov 27
PMID:Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5 A resolution. 981 28

Molybdenum is an essential element in plants and animals as a cofactor for enzymes. Molybdenum trioxide is used in metallurgical processes, in cosmetics as a pigment, and in contact lens solution, yet limited information is available on molybdenum genotoxicity. In the present study the micronucleus (MN) assay in human lymphocytes and mouse bone marrow and the dominant lethal assay in mice were used to assess the genotoxic effects of molybdenum salts in vitro and in vivo. Two salts of molybdenum were tested in whole blood cultures. Ammonium molybdate was more potent than sodium molybdate in causing a dose-dependent decrease in viability and replicative index and an increase in MN formation in binucleated lymphocytes (P < 0.001). A dose-response in both kinetochore-positive MN (caused by chromosome lagging) and kinetochore-negative MN (associated with chromosome breakage) was observed. Based on the results of a toxicity study of sodium molybdate, two doses, 200 and 400 mg/kg, were assessed in the bone marrow MN assay in mice (two i.p. injections 24 and 48 hr prior to euthanasia). A modest but statistically significant increase in MN frequency in polychromatic erythrocytes was observed (P < 0.05). The same treatment protocol was used to analyze dominant lethality. A dose-dependent increase in postimplantation loss represented mostly by early resorptions was observed the first week after treatment (P = 0.003). These preliminary data suggest that sodium molybdate induces dominant lethality at the postmeiotic stage of spermatogenesis. Overall, molybdenum salts produced moderately positive results both in vitro in human cells and in vivo in mice.
Environ Mol Mutagen 1998
PMID:Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo. 981 40

In Methanobacterium thermoautotrophicum, the fmdECB operon encoding the molybdenum formyl-methanofuran dehydrogenase is directly preceded by an open reading frame tfx predicted to encode a DNA binding protein. The 16.1 kDa protein has an N-terminal basic domain with a helix-turn-helix motif for DNA binding and a C-terminal acidic domain possibly for transcriptional activation. We report here on the DNA binding properties of the Tfx protein heterologously overproduced in Escherichia coli. Tfx was found to bind specifically to a DNA sequence downstream of the promoter of the fmdECB operon, as shown by electrophoretic mobility shift assays and DNase I footprint analysis. Northern blot hybridizations revealed that transcription of tfx is repressed during the growth of M. thermoautotrophicum in the presence of tung-state. Based on its structure and properties, the DNA binding protein Tfx is proposed to be a transcriptional regulator composed of a basic DNA binding domain and an acidic activation domain.
Mol Microbiol 1999 Jan
PMID:The DNA binding protein Tfx from Methanobacterium thermoautotrophicum: structure, DNA binding properties and transcriptional regulation. 1002 80

Molybdenum-containing enzymes of the hydroxylase class (such as xanthine dehydrogenase, aldehyde oxidase and nicotinate dehydrogenase) require a terminal sulphur atom attached to the molybdenum to hydroxylate their specific substrates. The transulphurylation reaction is carried out in Drosophila melanogaster by the product of the ma-I gene. In Aspergillus nidulans, the activity of the isofunctional and homologous HxB protein is needed in at least two different metabolic contexts, when the organism grows on purines and when it grows on nicotinate as nitrogen sources. We show here that the expression of the hxB gene is not constitutive. It is induced independently and additively by the inducers of the purine and of the nicotinate utilization pathways. Each of these induction pathways is affected independently by mutations in their cognate genes, uric acid induction by mutations in the UaY protein and nicotinate and 6-nicotinate induction by those in the hxnR/aplA complex. It is, in both metabolic contexts, exquisitely sensitive to nitrogen metabolite repression and highly dependent on the AreA GATA factor.
Mol Microbiol 1999 Feb
PMID:The hxB gene, necessary for the post-translational activation of purine hydroxylases in Aspergillus nidulans, is independently controlled by the purine utilization and the nicotinate utilization transcriptional activating systems. 1009 75


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