Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy.

Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo(VI) ion with each one MPT, Mo═O, Mo-O-, and Mo═S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo-OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.

Characterization of a novel Acinetobacter baumannii xanthine dehydrogenase expressed in Escherichia coli.

OBJECTIVE: To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications. RESULTS: The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40-45 °C, was stable under alkaline conditions (pH 7-11) and the half-inactivation temperature was 60 °C. The K m, turnover number and catalytic efficiency for xanthine were 25 µM, 69 s(-1) and 2.7 µM(-1) s(-1), respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized. CONCLUSION: The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

Structural and functional insights into the catalytic inactivity of the major fraction of buffalo milk xanthine oxidoreductase.

BACKGROUND: Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species. METHODS AND FINDINGS: Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m) of 0.11 sec(-1) µM(-1) of buffalo XO was 8-10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M(-1) cm(-1) suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD(+) entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum. CONCLUSION: A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.

Retinol oxidation to retinoic acid in human thyroid glandular cells.

Abstract Retinoic acid is regarded as the retinol metabolite that controls proliferation and differentiation of epithelial cells. In the present study, we investigated the potential role of xanthine dehydrogenase (XDH) in retinoic acid biosynthesis in human thyroid glandular cells (HTGC). In particular, we observed that cellular retinoids binding proteins (CRBPs) are also implicated in the biosynthetic pathway leading to retinoic acid formation in primary cultures of HTGC, as we have already reported for human mammary epithelial cells (HMEC). After partial protein purification, the enzyme responsible for retinoic acid biosynthesis was identified and quantified as XDH by immunoassay, by its ability to oxidize xanthine to uric acid and its sensitivity to the inhibitory effect of oxypurinol. The evidence of XDH-driven formation of retinoic acid in HTGC cultures further corroborates the potential role of XDH in retinoic acid biosynthesis in the epithelia.

FYX-051: a novel and potent hybrid-type inhibitor of xanthine oxidoreductase.

4-[5-(Pyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (FYX-051) is a potent inhibitor of bovine milk xanthine oxidoreductase (XOR). Steady-state kinetics study showed that it initially behaved as a competitive-type inhibitor with a K(i) value of 5.7 × 10(-9) M, then after a few minutes it formed a tight complex with XOR via a Mo-oxygen-carbon atom covalent linkage, as reported previously (Proc Natl Acad Sci USA 101:7931-7936, 2004). Thus, FYX-051 is a hybrid-type inhibitor exhibiting both structure- and mechanism-based inhibition. The FYX-051-XOR complex decomposed with a half-life of 20.4 h, but the enzyme activity did not fully recover. This was found to be caused by XOR-mediated conversion of FYX-051 to 4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (2-hydroxy-FYX-051), as well as formation of 6-hydroxy-4-[5-(2-hydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]pyridine-2-carbonitrile (dihydroxy-FYX-051) and 4-[5-(2,6-dihydroxypyridin-4-yl)-1H-1,2,4-triazol-3-yl]-6-hydroxypyridine-2-carbonitrile (trihydroxy-FYX-051) during prolonged incubation for up to 72 h. A distinct charge-transfer band was observed concomitantly with the formation of the trihydroxy-FYX-051-XOR complex. Crystallographic analysis of the charge-transfer complex indicated that a Mo-nitrogen-carbon bond was formed between molybdenum of XOR and the nitrile group of trihydroxy-FYX-051. FYX-051 showed a potent and long-lasting hypouricemic effect in a rat model of potassium oxonate-induced hyperuricemia, and it seems to be a promising candidate for the clinical treatment of hyperuricemia.

Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase.

Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Angstroms resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.

Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alphabeta)2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes.

Physicochemical and kinetic properties of purified sheep's milk xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) was purified for the first time from sheep's milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' milk enzymes and showed an A280/A450 ratio of 5.35 +/- 0.24, indicating a high degree of purity. Like milk XOR from other species, sheep's milk enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. Xanthine oxidase activity of purified sheep's milk XOR (0.69 +/- 0.04 micromole urate min(-1) mg(-1)) was low relative to that of the bovine milk enzyme (1.83 +/- 0.02 micromole urate min(-1) mg(-1)), but higher than those of human or goats' milk XOR. As in the latter 2 cases, the low activity of sheep's milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine milk enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's milk XOR was similar to that of enzymes purified from bovine, human, or goats' milk. The presence of desulpho-enzyme in sheep's milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.

High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa.

An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC. The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C. acidovorans. Co-expression of the xdhC gene is required for maximal level of functional expression. Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation. The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression. Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme. This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis. A method is also described to evaluate the suitability of P. aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.

Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

Factors involved in the assembly of a functional molybdopyranopterin center in recombinant Comamonas acidovorans xanthine dehydrogenase.

Previous work from this laboratory has shown that the spectral and functional properties of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans show some similarities to those of the well-characterized eukaryotic enzymes isolated from bovine milk and from chicken liver [Xiang, Q. & Edmondson, D.E. (1996) Biochemistry35, 5441-5450]. Therefore, this system was chosen to study the factors involved in the expression of functional recombinant enzyme in Escherichia coli to provide insights into the assembly of the functional Mo-pyranopterin center. Genes xdhA and xdhB (encoding the two known subunits of the native enzyme) and putative genes xprA and ssuABC were sequenced. Heterologous expression of the xdhAB genes in E. coli JM109(DE3) produced active enzyme. The Mo content was 0.11-0.16 mol per alphabeta protomer, while the Fe and FAD levels were at stoichiometries similar to that of the native enzyme. The XDH activity increased sixfold when the culture was grown under conditions of low aeration (6 L.min-1) as compared with high aeration (12 L.min-1). Co-expression of the xdhAB genes with the Pseudomonas aeruginosa PA1522 (xdhC) gene increased the level of Mo incorporated into the expressed enzyme to a 1 : 1 stoichiometry. Under these conditions, high levels of functional protein (2.284 U.mg-1 and 8.039 mg.L-1 of culture) were obtained independently of the level of culture aeration. Therefore, the assembly of a functional Mo-pyranopterin center in XDH requires the presence of a functional xdhC gene product. The purified, recombinant XDH shows spectral and kinetic properties identical to those of the native enzyme.

Purification and partial characterisation of camel milk xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.

Purification, crystallization and preliminary X-ray diffraction studies of xanthine dehydrogenase and xanthine oxidase isolated from bovine milk.

Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and the further oxidation of xanthine to uric acid. The enzyme is the target of the anti-gout drug allopurinol and its involvement in postischemic reperfusion injury is presently being defined. Each subunit of the homodimeric 290 kDa enzyme contains four cofactors: one Mo-pterin, two [2Fe-2S] clusters and one FAD. Both the dehydrogenase (XDH) and the proteolytically modified oxidase form (XO) of the enzyme from bovine milk have been crystallized. XO crystals belong to space group C222(1), with unit-cell parameters a = 116.3, b = 164.4, c = 153.2 A at room temperature and a = 117.8, b = 165.4, c = 154.5 A when flash-frozen. They allow data collection to 3.3 and 2.5 A, respectively. In addition, a data set was collected from frozen XDH crystals and processed to 2.1 A. These crystals belong to space group C2, with unit-cell parameters a = 169.9, b = 124.8, c = 148.6 A, beta = 90.9 degrees. The unit-cell volumes and Matthews parameters are similar for the two crystal forms. There is one monomer per asymmetric unit in the XO crystals and a complete native dimer per asymmetric unit in the XDH crystals.

Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation.

Xanthine oxidoreductase (XOR) has been purified from lactating mouse mammary tissue and its properties and developmental expression have been characterized. XOR was purified 80-fold in two steps using benzamidine-Sepharose affinity chromatography. The purified enzyme had a specific activity of 5.7 U/mg and an activity to flavin ratio of 192. SDS-polyacrylamide gel electrophoresis showed that it was composed of a single (150 kDa) band and N-terminal sequence analysis verified that it was intact mouse XOR. Isoelectric focusing showed that purified XOR was composed of three catalytically active, electrophoretic variants with pI values of 7.55, 7.65, and 7.70. The majority of the XOR activity in both pregnant and lactating mammary glands was shown to exist as NAD+-dependent dehydrogenase (XD form), while the enzyme in freshly obtained mouse milk exits as O2-dependent oxidase (XO form). The activity and protein levels of XOR selectively increased in mammary tissue during pregnancy and lactation. The time course of these increases was biphasic and correlated with the functional maturation of the mammary gland. These results indicate that XOR may have novel, mammary gland-specific functions, in addition to its role in purine metabolism.

Role of XDHC in Molybdenum cofactor insertion into xanthine dehydrogenase of Rhodobacter capsulatus.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is composed of two subunits, XDHA and XDHB. Immediately downstream of xdhB, a third gene was identified, designated xdhC, which is cotranscribed with xdhAB. Interposon mutagenesis revealed that the xdhC gene product is required for XDH activity. However, XDHC is not a subunit of active XDH, which forms an alpha2beta2 heterotetramer in R. capsulatus. It was shown that XDHC neither is a transcriptional regulator for xdh gene expression nor influences XDH stability. To analyze the function of XDHC for XDH in R. capsulatus, inactive XDH was purified from an xdhC mutant strain. Analysis of the molybdenum cofactor content of this enzyme demonstrated that in the absence of XDHC, no molybdopterin cofactor MPT is present in the XDHAB tetramer. In contrast, absorption spectra of inactive XDH isolated from the xdhC mutant revealed the presence of iron-sulfur clusters and flavin adenine dinucleotide, demonstrating that XDHC is not required for the insertion of these cofactors. The absence of MPT from XDH isolated from an xdhC mutant indicates that XDHC either acts as a specific MPT insertase or might be a specific chaperone facilitating the insertion of MPT and/or folding of XDH during or after cofactor insertion.

Modulation of renal xanthine oxidoreductase in aging: gene expression and reactive oxygen species generation.

Xanthine oxidoreductase (XD: xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes oxidation of hypoxathine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XOD). XOD is one of the major cellular sources of superoxide production and is well known as a causative factor in ischemia/reperfusion damage. At present, almost no information on the conversion status is available with respect to aging. In the present study, we investigated the effect of age on the XOD/XDH status and gene expression in the kidney. In addition, we assessed XOD-induced reactive oxygen species (ROS) using the dichlorofluoroscein (DCF) method. Our results show that XD activity gradually up to 18 months of age and then a slight decrease at 24 months of age. XDH activity showed increases up to 18 months of age, then decreased at 24 months of age. The conversion of XDH to XOD, assessed by changes in the ratios of XOD/(XOD+XDH), showed an age-related increase, which peaked at 24 months. Levels of XD protein and its mRNA paralleled to overall XD activity. ROS generation has tendency to increase with age. Our results suggest that the increased conversion of XDH to XOD observed with age may be an important contributing factor to the increased renal oxidative stress during aging.

Xanthine dehydrogenase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes.

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.