Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans*

Silke Leimkühler {ddagger} §, Rachael Hodson ¶, Graham N. George ¶ and K. V. Rajagopalan ||

From the {ddagger}Department of Plant Biology, Technical University Braunschweig, 38023 Braunschweig, Germany, Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Menlo Park, California 94025, and the ||Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, March 26, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an ({alpha}{beta})2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds. We have developed an efficient system for the recombinant expression of R. capsulatus XDH in Escherichia coli. The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase. However, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase. X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten LIII-edge have been used to probe the different metal coordinations of variant forms of the enzyme. Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Two different forms of XDH-R135C were purified, an active ({alpha}{beta})2 heterotetrameric form and an inactive ({alpha}{beta}) heterodimeric form. The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Xanthine dehydrogenase/oxidase (XDH1/XO) is a complex metallo-flavoprotein catalyzing the oxidative hydroxylation of purines, pyrimidines, pterines, and aldehyde substrates using NAD+ or molecular oxygen as electron acceptor. Mammalian XDH (EC 1.1.1.204 [EC] ) is a homodimer (290 kDa), with each subunit containing a single molybdenum cofactor (Moco) as well as two iron-sulfur clusters and an FAD molecule. Inherited XDH deficiency, referred to as xanthinuria I, is an autosomal recessive disease that is characterized by hyperuricemia, hyperuricosuria, and xanthinuria and is based on base pair exchanges in the structural gene for XDH (1). The affected individuals may develop urinary tract calculi, acute renal failure, or myositis, because of tissue deposition of xanthine, although some subjects with homozygous xanthinuria remain asymptomatic (1). Several mutations in the human XDH gene in patients with classical xanthinuria have been reported (2, 3, 4).

In their native form, mammalian xanthine oxidizing enzymes exist as dehydrogenases that transfer electrons to NAD but can undergo a dehydrogenase to oxidase conversion by proteolytic cleavage or by oxidation, with the concomitant loss of the ability to use NAD+ as electron acceptor, and nearly 10-fold increase in the oxidase activity. The crystal structures of bovine milk XDH in both the dehydrogenase and oxidase forms have been solved at 2.1 and 2.5 Å resolution, respectively (5). It was shown that during the conversion of XDH to XO, a specific structural rearrangement blocks access of NAD+ to its binding site near the FAD moiety and changes the electrostatic environment of the bound FAD, reflecting the switch of electron acceptor specificity observed for the two forms of this enzyme.

A well characterized prokaryotic XDH with similar activity to the mammalian enzymes is the enzyme isolated from the phototrophic purple bacterium Rhodobacter capsulatus (6) Despite the similarities to the mammalian XDH enzymes, R. capsulatus XDH, like avian XDH, is isolated with high reactivity toward NAD+ and low reactivity toward oxygen as electron acceptor and does not undergo the conversion to the oxidase form. R. capsulatus XDH is a cytoplasmic enzyme with an ({alpha}{beta})2 heterotetrameric structure and a molecular mass of 275 kDa. The enzyme is encoded by two genes, xdhA and xdhB, with the FeS clusters and FAD bound to the XDHA subunit and Moco bound to the XDHB subunit. A third protein, designated XDHC, was shown to be essential for XDH activity in R. capsulatus but was not found to be a subunit of active XDH (7). Because XDH isolated from an R. capsulatus xdhC mutant strain was shown to be Moco-deficient, the role of XDHC for XDH activity was proposed to be related to the insertion of Moco into XDHB during the assembly of XDH. The crystal structure of R. capsulatus XDH has been solved recently at 2.7 Å resolution (8), showing that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. However, the two structures differ in important details, including the regions necessary for XDH to XO conversion. Because of the high structural similarities of the mammalian XDH/XO and R. capsulatus XDH, the bacterial enzyme is a good model system for studying the mechanism of the enzyme and can be used for the generation of site-specific mutants.

In this report we describe the establishment of a recombinant expression system for R. capsulatus XDH in Escherichia coli cells, as well as the biochemical characterization of the recombinant enzyme. The recombinant protein resembles bovine XO in its absorption spectrum and its wide range of substrate specificity but is about 5 times more active compared with the mammalian enzymes. The enzyme has been also examined using x-ray absorption and EPR spectroscopy. EPR analysis of the FeS centers showed two characteristic EPR signals for FeSI and FeSII seen with all members of the xanthine oxidase family (9). In addition, x-ray absorption spectroscopy at the molybdenum and tungsten K-edge/LIII-edge, respectively, have been used to compare the active sites of the native and tungsten-substituted enzymes. Because the heterologous expression system of R. capsulatus XDH in E. coli allows purification of sufficient enzyme for structural and kinetic studies, it is a useful tool for the generation of site-specific mutants for structural and biochemical studies. By using this system, the structural bases of xanthinuria I can be examined that will help to explain different symptoms of the disease. Based on a mutation identified in a patient suffering from xanthinuria I, arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Purified heterologously expressed XDH-R135C mutant contained two different protein forms in almost equal quantities, an active ({alpha}{beta})2 heterotetramer and an inactive ({alpha}{beta}) heterodimer.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Media, and Growth Conditions—The E. coli TP1000 mutant strain used in this study is a derivative of MC4100 with a kanamycin cassette inserted in the mobAB gene region (10). The E. coli RK5200 strain is a chlA::Mu cts mutant of RK4353 (11). E. coli BL21(DE3) cells and pET15b were obtained from Novagen. The expression plasmid pTrcHis used for heterologous expression of R. capsulatus XDH in E. coli was described previously (12). For expression of pET15b-based plasmids, the {lambda} DE3 lysogenization kit from Novagen was used to integrate the gene for T7 RNA polymerase into the E. coli strain RK4353 (11). Cell strains containing expression plasmids were grown aerobically at 30 °C in LB medium in the presence of 150 µg/ml ampicillin. Phenyl-Sepharose resin was purchased from Amersham Bio-science; Q-Sepharose resin was purchased from Sigma, and Ni-NTA-agarose was purchased from Qiagen.

Cloning, Expression, and Purification of Native XDH and Protein Variants—The genes encoding R. capsulatus XDH, designated xdhABC, were isolated from the R. capsulatus genome as described previously (6). The published gene sequence (6) was used to design primers that permitted cloning into the NdeI and HindIII sites of the expression vector pTrcHis (12). The resulting plasmid, designated pSL207, contains the xdh genes with a His6 tag fused to the N terminus of XDHA. For heterologous expression in E. coli, pSL207 was transformed into E. coli TP1000 cells (10), containing a deletion in the mobAB genes responsible for Moco dinucleotide formation. The enzyme was expressed in 500-ml cultures of TP1000 cells carrying plasmid pSL207 grown at 30 °C in LB medium supplemented with 150 µg/ml ampicillin, 1 mM molybdate, and 0.02 mM isopropyl-{beta}-D-thiogalactopyranoside until the OD = 1. This culture was then transferred to a bottle containing 8 liters of supplemented LB medium and subsequently grown at 30 °C for 18–20 h. Cells were harvested by centrifugation at 5000 x g. The cell pellet was resuspended in 8 volumes of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, and cell lysis was achieved by several passages through a French press. After addition of DNase I, the lysate was incubated for 30 min. After centrifugation at 17,000 x g for 25 min, imidazole was added to the supernatant to a final concentration of 10 mM. The supernatant was mixed with 2 ml of Ni2+-nitrilotriacetic agarose (Qiagen) per liter of cell growth, and the slurry was equilibrated with gentle stirring at 4 °C for 30 min. The slurry was poured into a column, and the resin was washed with 2 column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, followed by a wash with 10 column volumes of the same buffer with 20 mM imidazole. His-tagged XDH was eluted with 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing XDH were combined and dialyzed against 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, pH 7.5. The dialyzed sample was applied to a Q-Sepharose fast protein liquid chromatography column and eluted with a linear gradient of 0–250 mM NaCl. To the pool of fractions containing XDH, 15% ammonium sulfate was added, and the protein was then applied to a phenyl-Sepharose column equilibrated with 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, 15% ammonium sulfate, pH 7.5. XDH was eluted from the column with a linear gradient of 15 to 0% ammonium sulfate. During purification, fractions were monitored using SDS-PAGE, whereas enzyme activity was measured spectrophotometrically as described earlier (6). The yield of protein was about 12.5 mg/liter of E. coli culture. To obtain the tungsten-substituted form of XDH, the same growth conditions and purification procedures were used with the exception that the LB medium was supplemented with 5 mM tungstate instead of molybdate. For expression of xdhAB in the absence of xdhC, the NdeI-HindIII fragment from pSL207 was cloned into pET15b, and an XhoI fragment containing the coding region for xdhC was deleted from this plasmid by partial XhoI digest to generate plasmid pSL195. For expression, pSL195 was transformed into RK4353(DE3) cells and was grown under the same conditions as described above, and the protein was purified as described earlier. For expression of XDHA and XDHB subunits individually, primers were designed that permitted cloning of the xdhA coding sequence and the xdhBC coding sequence into the NdeI site of pET15b after PCR. These constructs resulted in an N-terminal His6 fusion of XDHA and XDHB, respectively. The expression plasmids for xdhA and xdhBC were designated pSL180 and pSL178, respectively. For expression, E. coli RK4353(DE3) cells were transformed with pSL178 and pSL180, and 6x 1 liter of transformed RK4353(DE3) cells were grown under the same conditions used for wild-type XDH as described above. The cell pellet was resuspended in 8 volumes of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, and cell lysis was achieved by several passages through a French press.

Site-directed Mutagenesis of XDH and Purification of XDH-R135C— The transformer site-directed mutagenesis kit (Clontech) was employed for the generation of the single amino acid substitution R135C in R. capsulatus XDH. The nucleotide sequence substitution was verified by automated sequencing. To obtain the R135C variant for XDH the same growth conditions were used as described above. After chromatography on Q-Sepharose, the protein was gel-filtered on a Superose 12 column (Amersham Bioscience) using 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, 100 mM NaCl, pH 7.5. Determination of the molecular weight of XDH-R135C variants was carried out using a Sephadex 200 (Amersham Biosciences) gel filtration column in 50 mM Tris, 1 mM EDTA, 200 mM NaCl, pH 7.5.

UV-visible Absorption Spectra—Absorption spectroscopy was carried out using a Shimadzu UV-2101 PC spectrophotometer. All spectra were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5, using a 1-ml cuvette. For comparison spectra, bovine milk xanthine oxidase used was purified as described earlier (13).

Molybdopterin (MPT) Analysis by Generation of Form A (Dephospho)—To determine the amount of Moco present in XDH, the purified proteins were subjected to a procedure that converts MPT to the stable oxidized fluorescent degradation product form A by treatment with iodine at pH 2.5 at room temperature (14). After treatment with alkaline phosphatase, dephospho form A was identified by HPLC analysis with an Alltech C18 HPLC column in 50 mM ammonium acetate, 10% methanol at a flow rate of 1 ml/min. Form A was assayed by monitoring its fluorescence with an excitation at 297 nm and emission at 440 nm. All HPLC analyses were performed using the Hewlett-Packard 1090 solvent delivery system, and the eluted material was monitored either for absorbance using a Hewlett-Packard 1040A diode array detector or for fluorescence using a Hewlett-Packard 1046 fluorescence detector.

Trypsin Digestion of R. capsulatus Xanthine Dehydrogenase—Purified R. capsulatus xanthine dehydrogenase (1 mg/ml) was incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (2% w/v) in 50 mM Tris, 1 mM EDTA, pH 7.5, at room temperature for 1 h prior to PAGE on 10% polyacrylamide gels under denaturing conditions.

PAGE—For PAGE, protein samples were heated at 95 °C in buffer containing 2% SDS and 5% {beta}-mercaptoethanol. Perfect protein markers from Novagen were used as molecular mass standard. Electrophoresis was carried out on 7.5 or 10% polyacrylamide ready gels (Bio-Rad), and the gels were stained with Coomassie Brilliant Blue R (Sigma).

Enzyme Assays—All xanthine dehydrogenase assays were carried out using a Shimadzu UV-2101 PC spectrophotometer. Assays were performed in 50 mM Tris, 1 mM EDTA, pH 7.5, at room temperature in a final volume of 1 ml. Routine assay mixtures contained 100 µM xanthine. Enzyme activity was assayed spectrophotometrically at 295 nm when xanthine or hypoxanthine was used as substrate or at 600 nm with 10 µM dichloroindophenol as electron acceptor when purine, pterin, acetaldehyde, glyceraldehyde, or 1-methylnicotinamide was used as substrate. The inhibition of enzyme activity by 100 µM allopurinol was analyzed in the presence of 100 µM xanthine as substrate, monitoring activity at 295 nm. Assays containing R. capsulatus xanthine dehydrogenase were recorded in the presence of 100 µM NAD as electron acceptor, whereas for bovine milk xanthine oxidase O2 was used as electron acceptor.

Cyanide Inactivation and Resulfuration of R. capsulatus XDH— R. capsulatus XDH (5 mg/ml) was incubated with 50 mM KCN in 50 mM Tris, 1 mM EDTA, pH 7.5, for 18 h at 4 °C. Excess cyanide was removed from the mixture by gel filtration using a PD10 column equilibrated in 50 mM Tris, 1 mM EDTA, pH 7.5. Desulfo-XDH was resulfurated by addition of 5 µl of 0.1 M dithionite and 1 µl of 0.5 M Na2S under anaerobic conditions using a Coy anaerobic chamber. After addition of Na2S and dithionite, the protein was incubated for 30 min at room temperature before excess sulfide was removed from the mixture by gel filtration.

Analytical Methods—Phosphate was quantitated as described by Ames (15); iron content was determined as described by Fish (16), and the FAD content was quantitated as described by Faeder and Siegel (17). For quantification of total protein concentrations, the Pierce BCA assay was used as described in the manufacturer's protocol, with bovine serum albumin as the standard.

Electron Paramagnetic Resonance Spectroscopy—Spectra were recorded on JEOL RE1X or Bruker ER300 spectrometers equipped with an Oxford Instruments ESR 9 liquid helium cryostat. Samples were prepared as frozen solutions (typically ~0.1 mM enzyme) in 3-mm diameter quartz tubes. Magnetic field was calibrated using a diphenylpicrylhyrdazyl standard, assuming g = 2.0037, and microwave frequency was determined using a Hewlett-Packard frequency counter.

X-ray Absorption Spectroscopy—Measurements were carried out at the Stanford Synchrotron Radiation Laboratory with the SPEAR storage ring containing 60–100 mA at 3.0 GeV, on beamlines 9-3 and 7-3 operating with a wiggler field of 2.0 and 1.8 T, respectively, and using a Si(220) double crystal monochromator. Beamline 9-3 is equipped with a rhodium-coated collimating mirror upstream of the monochromator, and a bent cylindrical rhodium-coated focusing mirror downstream of the monochromator. Harmonic rejection was accomplished by setting the cut-off energy of the focusing mirror to 11, 13, or 23 keV for iron K-edge tungsten LIII edge and molybdenum K-edge measurements, respectively. The incident x-ray intensity was monitored using nitrogen-(for tungsten LIII experiments) or argon (for molybdenum K-edge experiments)-filled ionization chambers. The x-ray absorption was measured as the x-ray K{alpha} for iron and molybdenum or L{alpha}1 for tungsten fluorescence excitation spectrum using an array of 30 germanium intrinsic detectors (18). During data collection, samples were maintained at a temperature of ~10 K using a liquid helium flow cryostat. For each sample between 6 and 10 35-min scans were accumulated, and the absorption of a standard metal foil was measured simultaneously by transmittance. The energy was calibrated with reference to the lowest energy inflection points of the metal foil standards, which were assumed to be 20,003.9, 10,207.0 and 7111.3 eV, for the molybdenum K-, tungsten LIII-, and iron K-edges, respectively.

The extended x-ray absorption fine structure (EXAFS) oscillations {chi}(k) were quantitatively analyzed by curve-fitting using the EXAFSPAK suite of computer programs2 (19) as described by George et al. (20), using ab initio theoretical phase and amplitude functions calculated using the program FEFF version 8.2 (21).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterologous Expression and Purification of R. capsulatus XDH in E. coli—The conditions for the heterologous expression of R. capsulatus XDH described here were the same as reported for the expression of human sulfite oxidase in E. coli (12). The protein was purified from an 8-liter E. coli culture by Ni-NTA chromatography, followed by chromatography on Q-Sepharose and phenyl-Sepharose (Table I). The purified protein displayed two discrete bands on Coomassie Brilliant Blue R-stained SDS gels (Fig. 1), corresponding to the XDHA (50 kDa) and XDHB (85 kDa) subunits of the protein (6). After coexpression of xdhAB with the xdhC gene in E. coli, no subunit corresponding to XDHC was identified after purification (Fig. 1). This result is in agreement with the data obtained after homologous expression of xanthine dehydrogenase in R. capsulatus (7), showing that XDHC acts as a specific chaperone for XDH maturation but is itself not a subunit of the active enzyme.


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TABLE I
Purification procedure of recombinant R. capsulatus XDH after expression in E. coli TP1000 cells

 


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FIG. 1.
Purification of R. capsulatus XDH. 7.5% SDS-PAGE analysis of R. capsulatus XDH after different purification stages. Each lane contained ~15 µg of protein.

 

Characterization of the Role of XDHC for the Maturation of R. capsulatus Xanthine Dehydrogenase in E. coli—Leimkühler and Klipp (7) reported that in R. capsulatus XDHC is required for the insertion of Moco into the XDH tetramer, perhaps serving as a specific chaperone facilitating the insertion of the cofactor into the apoprotein. To determine whether XDHC is also required for the maturation of R. capsulatus XDH in E. coli, R. capsulatus XDH was expressed in E. coli RK4353-(DE3) cells from plasmid pSL195 carrying the xdhAB genes only (see ";Experimental Procedures"). After expression, the protein was purified as described above. Analysis of enzyme activity showed that XDH expressed in the absence of xdhC was inactive. Surprisingly, analysis of the cofactor content of the protein showed that the protein contained Moco. As shown in Fig. 2, after conversion of protein-bound Moco to its oxidized fluorescent degradation product form A, HPLC analysis revealed the same amount of cofactor in XDH in the presence or absence of xdhC during expression. Thus, the role of XDHC in the maturation of R. capsulatus XDH in E. coli is different from that observed for homologous expression of XDH in an R. capsulatus xdhC mutant strain.



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FIG. 2.
Influence of XDHC on the cofactor insertion into R. capsulatus XDH after expression in E. coli. Analysis of fluorescent derivatives of Moco from R. capsulatus XDH expressed in the presence of the xdhC from plasmid pSL207 (A) or in the absence of xdhC from plasmid pSL195 (B). 20 µM purified R. capsulatus XDH was used for the analyses.

 

Identification of a Cyanolyzable Sulfur Ligand in R. capsulatus Xanthine Dehydrogenase—Xanthine dehydrogenase, xanthine oxidase, and aldehyde oxidase belong to a family of molydoenzymes characterized by a non-protein sulfur ligand to the molybdenum atom. This terminal sulfur atom is essential for enzyme activity and is released as thiocyanate upon inactivation of the enzyme with cyanide. Most preparations of xanthine dehydrogenase/oxidase from eukaryotic sources contain varying fractions of demolybdo proteins and also contain desulfo molecules deficient in the cyanolyzable sulfur. However, the desulfo molecules can be activated by treatment with sulfide and dithionite under anaerobic conditions (22).

As shown in Table II, R. capsulatus XDH was inactivated by incubation with 2.5 mM cyanide at room temperature. After removal of excess cyanide, reactivation of the inactive enzyme required both sulfide and dithionite. In contrast, the activity of enzyme not treated with CN-was not enhanced by sulfide/dithionite treatment. These data firmly establish the presence of the terminal sulfur ligand in R. capsulatus XDH. To determine whether the Moco present in XDH expressed in the absence of xdhC contains the desulfo form of the cofactor or the demolybdo form, purified XDH expressed in the presence or absence of xdhC was subjected to treatment with sulfide and dithionite. As shown in Table II, XDH expressed in the absence of xdhC was activated by sulfide/dithionite treatment to the same extent as XDH expressed in the presence of xdhC and desulfurated by cyanide treatment. This observation shows that during heterologous expression of R. capsulatus XDH in E. coli, R. capsulatus XDHC is required for the sulfuration of Moco but not for the insertion of Moco into XDH, a situation that is different from the requirement of XDHC for cofactor insertion into XDH in the native host R. capsulatus (7).


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TABLE II
Influence of sulfide / dithionite on active R. capsulatus XDH, cyanide-inactivated XDH, and inactive XDH expressed in the absence of xdhC

 

Separate Expression of XDHA and XDHB Subunits—Because XDHB contains only Moco, its expression in the absence of XDHA could provide the means for spectroscopic characterization of the XDH-type molybdenum center without interference from the FAD and Fe/S chromophores. It was therefore of interest to express the XDHA and XDHB subunits of R. capsulatus XDH individually. For this purpose, XDHA and XDHB were cloned into the pET15b vector (see ";Experimental Procedures"), yielding an N-terminal His6 fusion at each subunit. To ensure the insertion of sulfurated Moco into the single XDHB subunit, the xdhB gene was coexpressed with xdhC (see ";Experimental Procedures"). However, even after varying the expression conditions in E. coli RK4353(DE3) cells, attempts to purify single R. capsulatus XDHA or XDHB subunit were unsuccessful (data not shown). These results are consistent with mutant studies in R. capsulatus, where it was impossible to detect the XDHA or the XDHB subunit in xdhB or xdhA mutants, respectively, by immunoblot analysis (7).

Comparison of Substrate Specificities of R. capsulatus XDH and Bovine Milk XO—As shown in Table III, both enzymes show the same broad spectrum of substrate specificities. The catalytic activity is expressed as AFR and is about 14 times higher for R. capsulatus XDH as compared with bovine milk XO. However, the AFR for completely active XO is reported to be 200, showing that XO used in these assays was only 42% active. Still, the AFR of R. capsulatus XDH is at least 5 times higher than that of fully active XO, showing a much higher turnover number for R. capsulatus XDH.


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TABLE III
Comparison of substrate specificities for R. capsulatus XDH and bovine milk XO

 

Cofactor Analysis of R. capsulatus XDH—The preparation of R. capsulatus XDH contained 0.9 FAD, 2.8 phosphate, and 3.7 iron per XDHAB subunit (Table IV), showing that the purified recombinant protein contains a full complement of cofactors. The Moco content of the protein was estimated by form A analysis to be 70% in comparison to bovine milk XO (Fig. 3). The absorption spectrum of recombinant R. capsulatus XDH (Fig. 4) shows the presence of Fe/S and FAD prosthetic groups. The ratios of A280/A465 of 5.0 and of A465/A550 of 3.0 (Table IV) are similar to those of other metalloflavoproteins, corresponding to a FAD to iron ratio of 1:4. The absorption maximum for R. capsulatus XDH is at 465 nm somewhat red-shifted compared with the spectrum of bovine XO (Fig. 4). This difference is probably due to different environments of FAD in R. capsulatus XDH and bovine XO. Fig. 5 shows differences in the absorption spectra of cyanide-treated desulfo-XDH in comparison to the enzyme not treated with cyanide and to XDH without bound Moco.


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TABLE IV
Spectral properties, phosphate, FAD, and iron content of recombinant R. capsulatus XDH

 


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FIG. 3.
Analysis of fluorescent derivatives of Moco from R. capsulatus XDH and bovine milk XO. HPLC elution profiles of form A isolated from 20 µM R. capsulatus XDH (A), and 20 µM bovine milk XO (B).

 


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FIG. 4.
UV-visible absorption spectra of R. capsulatus XDH and bovine milk XO. Spectra of 1 mg of R. capsulatus XDH (solid line) and 1 mg of bovine milk XO (dashed line). Spectra were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5.

 


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FIG. 5.
UV-visible absorption spectra of sulfurated R. capsulatus XDH, desulfo-XDH, and XDH without Moco. Spectra of 0.85 mg of R. capsulatus XDH as purified (solid line), 0.85 mg of cyanide-treated desulfo-XDH (dashed line) and 0.85 mg of XDH without Moco (dashed dotted line) purified from the Moco-deficient E. coli strain RK5200. Spectra were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5.

 

Proteolytic Cleavage of R. capsulatus XDH—All animal xanthine dehydrogenases with the exception of avian liver XDH exist in vivo as NAD-dependent (type D) enzymes. Most of the type D enzyme is converted to an O2-dependent type (type O) during purification (23). This conversion occurs either reversibly through oxidation of sulfhydryl groups (23) or irreversibly through proteolysis of the enzyme molecule (24). After proteolytic treatment of bovine milk XDH, the enzyme is irreversibly converted to the O-type consisting of three tightly associated fragments with molecular weights of 90,000, 60,000, and 30,000, respectively. Because R. capsulatus XDH showed only very low activity with molecular oxygen (2%, data not shown), it seems that the enzyme, like chicken liver XDH, is a true dehydrogenase that is not converted to the oxidase form during purification. To test the effect of proteolysis on R. capsulatus XDH, the enzyme was subjected to a treatment with trypsin (see ";Experimental Procedures"). As shown in Fig. 6, when R. capsulatus XDH is treated with trypsin, the XDHB subunit it is cleaved into four fragments with molecular weights of 35,000, 30,000, 20,000 and 15,000, whereas the XDHA subunit remains unchanged. However, the quaternary structure and the activity of the enzyme remain unaltered, showing no increase in the activity with molecular oxygen as electron acceptor (data not shown).



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FIG. 6.
SDS-PAGE analysis of trypsin-treated R. capsulatus XDH. 10% SDS-PAGE of untreated and trypsin-treated R. capsulatus XDH. XDH (1 mg/ml) was incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (2% w/v) in 50 mM Tris, 1 mM EDTA, pH 7.5, for 1 h at room temperature. Each lane contained ~15 µg of protein.

 

Tungsten Derivative of R. capsulatus XDH—To determine whether tungsten can be incorporated in lieu of molybdenum into R. capsulatus XDH, cells were grown in the presence of 5 mM tungstate. Expression and purification conditions were the same as described above. The Moco content of the tungsten derivative of XDH was shown to be 50% compared with XDH containing molybdenum as the bound metal (Fig. 7). As expected, the tungsten derivative is not active with xanthine or hypoxanthine as substrate, and the absorption spectra show that the enzyme is not reduced with xanthine but is fully reduced with dithionite (Fig. 8).



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FIG. 7.
Fluorescence spectra of form A derivatives from Moco of molybdenum-XDH and the tungsten-XDH derivative. Moco was released from 70 µM molybdenum (Mo)-XDH and 70 µM tungsten (W)-XDH and converted to its oxidized fluorescent degradation product form A by treatment with acidic iodine. Fluorescence spectra were recorded using an Aminco-Bowman SPF spectrofluorometer, with the emission wavelength set at 460 nm for excitation spectra and the excitation wavelength set at 380 nm for emission spectra.

 


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FIG. 8.
UV-visible absorption spectra of the R. capsulatus tungsten-XDH derivative. Spectra of 1.2 mg of the air-oxidized R. capsulatus tungsten-XDH (solid line), of 1.2 mg of tungsten-XDH incubated with 10 mM xanthine (dashed line) and of 1.2 mg of tungsten-XDH reduced with dithionite (dashed dotted line). Spectra were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5, under aerobic conditions.

 

Investigation of the Fe/S Clusters of R. capsulatus XDH Using Electron Paramagnetic Resonance and X-ray Absorption Spectroscopy—Because of the differences in UV-visible spectra of bovine XO and R. capsulatus XDH, the EPR and x-ray absorption spectroscopic properties of the FeS clusters were investigated. Fig. 9A shows the EPR spectra of the FeS clusters of dithionite-reduced R. capsulatus XDH, together with computer simulations. For all members of the xanthine oxidase family of enzymes that have been described to date, two characteristic iron-sulfur EPR signals are observed, designated as FeSI and FeSII (25). FeSI has EPR properties similar to those of many other [Fe2S2]+ proteins, being fully developed at relatively high temperatures (e.g. 45 K), and a gav close to 1.95. FeSII, on the other hand, has unusual EPR properties for a [Fe2S2]+ species, being only observed at much lower temperatures, having unusually broad line widths and an unusually high gav close to 2.0. R. capsulatus XDH clearly shows FeSI and FeSII EPR signals, with the notable difference (from bovine XO) that FeSI of R. capsulatus XDH is axial (g{perp},|| = 1.922, 2.022), rather than rhombic (XO gx,y,z = 1.894, 1.932, 2.022, gav = 1.949), and possesses considerably sharper line widths, although with a very similar gav of 1.952. FeSII is also sharper than the corresponding xanthine oxidase signal and also has subtly different g values (gx,y,z = 1.896, 1.971, 2.073, compared with gx,y,z = 1.902, 1.991, 2.110 for XO), with different gav 1.980 for XDH compared with 2.001 for XO. We note that part of the latter discrepancy may be due to the large line widths for gx in xanthine oxidase, which might have caused significant errors in the determination of this parameter.



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FIG. 9.
FeS EPR spectra and iron K-edge EXAFS of R. capsulatus XDH. A, low temperature EPR spectra of R. capsulatus XDH at 9.05515 GHz. The de-Moco form of XDH was used to prevent interference from Mo(V). Enzyme (~0.1 mM) was reduced with 2 mM sodium dithionite solution at pH 7.5 (50 mM Tris, 1 mM EDTA) under anaerobic conditions. The FeSI signal was recorded at 42 K and that of both FeSI and FeSII at 18 K. In both cases 0.1 milliwatt of applied power and a modulation amplitude of 0.5 mT were used. Parameters derived from EPR simulations were gx,y,z = 1.9217, 1.9217, 2.0222 for Fe/S I, and gx,y,z = 1.8964, 1.9711, 2.0730 for Fe/S II. Pseudo-Voigt peak shapes were employed with half-line widths gx,y,z of 1.25, 1.25, 1.12 mT for FeSI, and gx,y,z of 1.6, 1.5, and 1.7 mT for FeSII. The simulation of the FeSI + FeSII spectrum was computed assuming equal spin intensities for FeSI and FeSII. B, iron K-edge EXAFS of R. capsulatus XDH. The EXAFS Fourier transform (Fe-S phase-corrected) and EXAFS oscillations (inset) are shown as solid lines, with the results of curve-fitting analysis as dashed lines. The parameters derived from curve-fitting analysis are summarized in Table V.

 


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TABLE V
EXAFS curve-fitting results

 
The iron K-edge EXAFS spectrum and corresponding Fourier transform of oxidized XDH are shown in Fig. 9B. Clearly defined Fe-S and Fe···Fe interactions are manifest in the Fourier transform peaks at 2.3 and 2.7 Å, respectively. EXAFS curve-fitting analysis indicates in Fe-S and Fe···Fe distances of 2.26 and 2.72 Å, respectively, which can be compared with the analogous inter-atomic distances derived from oxidized milk xanthine oxidase of 2.25 and 2.71 Å (data not shown). These represent the average distances for both FeSI and FeSII, and no resolved differences indicating two structural types of cluster are observed. Furthermore, the structural similarity between FeSI and FeSII is indicated by the relatively low Debye-Waller factors (of 0.0057 and 0.0043 Å2, for Fe-S and Fe···Fe, respectively). The Debye-Waller factors contain both vibrational and static components, and the size of these is close to the expected vibrational lower limit, suggesting that the core structure of the two FeS clusters is very similar. The iron K near-edge spectra (not illustrated) are typical of a [Fe2S2]2+ cluster and are essentially superimposable with the spectra of bovine XO and with 2Fe ferredoxins.3

Molybdenum K-edge and Tungsten LIII-edge X-ray Absorption Spectroscopy—In order to compare the active sites of the molybdenum- and tungsten-substituted enzymes, we measured the molybdenum K and tungsten LIII-edge EXAFS spectra of samples of these forms of the enzyme. Fig. 10 shows the EXAFS data of both cyanolyzed molybdenum-XDH and as-isolated tungsten-XDH (which was mostly in the desulfo form), together with the results of curve-fitting analysis. The Fourier transforms can be seen to be very similar, definitively indicating a close structural relationship between the molybdenum- and tungsten-containing active sites. The quantitative structural parameters derived from EXAFS curve-fitting analysis are summarized in Table V. The EXAFS spectrum of fully active enzyme proved more elusive as the protein tended to lose activity during preparation at the high concentrations required for XAS. The Mo(IV) alloxanthine-bound form, on the other hand, could be prepared in quantity, and the EXAFS data for this are shown in Fig. 11, with the curve-fitting results summarized in Table V. The XDH alloxanthine complex is very similar to that of bovine XO described by Bray and co-workers (26). The metal is coordinated by one oxo-ligand at 1.71 Å, three thiolates at 2.37 Å, and one oxygen or nitrogen at 2.01 Å (Table V). We note that EXAFS is not normally capable of distinguishing between oxygen and nitrogen, and essentially equivalent fits were obtained with oxygen in place of nitrogen or vice versa. The fit was marginally better with Mo-N (error 0.227 versus 0.235); however, it is not possible to assign these ligands unambiguously based on EXAFS data alone. Two of the three sulfurs can be assumed to come from the Moco dithiolene, whereas the third is likely a Mo-SH ligand arising from protonation of the Mo=S ligand of oxidized enzyme (a Mo=S ligand would be expected to have a bond length of about 2.16 Å). The recent crystallography of the XDH-alloxanthine complex suggests a mono-oxo site with the N8 atom of alloxanthine coordinated directly to molybdenum and an axial Mo-S, which probably is actually a Mo-SH. The EXAFS and the crystallography are thus in excellent agreement.



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FIG. 10.
Comparison of the molybdenum- and tungsten-desulfo XDH EXAFS data. The EXAFS Fourier transforms (phase-corrected for oxygen backscattering) and EXAFS oscillations (insets) for both molybdenum (Mo)- and tungsten (W)-XDH are shown in the upper and lower panels, respectively. The solid lines denote experimental data and the dashed lines the results of curve-fitting analysis. The parameters derived from curve-fitting analysis are summarized in Table V.

 


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FIG. 11.
EXAFS data of the molybdenum-XDH alloxanthine complex. The EXAFS Fourier transform (phase-corrected for Mo-S backscattering) and the EXAFS oscillations (inset) are shown. The solid lines denote experimental data and the dashed lines the results of curve-fitting analysis. The parameters derived from curve-fitting analysis are summarized in Table V.

 

Characterization of R. capsulatus XDH-R135C Variant Generated after a Mutation Identified in a Xanthinuria I Patient—In a case of xanthinuria I exhibiting hyperuricemia, a point mutation in the structural gene for human XDH was identified (4), revealing the exchange of arginine 149 to a cysteine, and the patient was diagnosed as homozygous for this mutation. The XDH protein was still present in the patient, showing that the mutation did not influence the stability of the protein (4). Arg-149 is a conserved arginine residue in XDH proteins, being located in-between two conserved cysteine residues involved in the formation of the FeSI cluster. To identify the structural basis of the R149C mutation leading to the inactivation of XDH, we generated the corresponding R135C amino acid substitution in R. capsulatus XDH by site-directed mutagenesis (see ";Experimental Procedures"). The XDH-R135C variant was expressed and purified from the E. coli TP1000 strain by Ni-NTA chromatography, Q-Sepharose, and size exclusion chromatography. After size exclusion chromatography, two forms of the XDH-R135C variant were identified, which were well separated during size exclusion chromatography (Fig. 12A). One form of the XDH-R135C eluted at the same time as the native protein (data not shown), corresponding to ({alpha}{beta})2 heterotetramer. The other form displayed a molecular weight expected of an ({alpha}{beta}) heterodimer. The UV-visible absorption spectra of the two protein forms in comparison to native XDH are shown in Fig. 12B. The absorbance spectrum of the heterotetrameric form was almost identical to that of native XDH (Fig. 12B). In contrast, the heterodimer displayed diminished absorbance in the visible region, with a shift in the absorbance peak from 465 to 450 nm (Fig. 12B). The absorbance ratio A450/A550 of 4.4 of the heterodimeric form is higher than for native XDH (Table VI), suggesting a decreased FeS cluster content. The active variant showed 90% of the activity of native XDH, whereas the heterodimeric form was virtually inactive (Table VI). The Moco content of the two variant mutant forms correlated with their activities (Table VI).



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FIG. 12.
Size exclusion chromatography and UV-visible absorption spectra of R. capsulatus XDH-R135C variant. A, chromatogram after size exclusion chromatography (Sephadex 200, Amersham Biosciences) of 0.3 mg of active and 0.3 mg of inactive protein pools of XDH-R135C variant. B, UV-visible absorption spectra of 1.9 mg of active (dashed line) and inactive (dashed dotted line) XDH-R135C variants in comparison to wild-type XDH (solid line).

 

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TABLE VI
Enzyme activities, spectral properties, Moco, and Iron content of R. capsulatus XDH-R135C variant

 

Investigation of the Fe/S Clusters of R. capsulatus XDH-R135C Variant Using EPR—To delineate further the effects of the R135C mutation on FeSI or FeSII, EPR spectroscopy was carried out on native XDH and the active heterotetrameric form of the mutant. The FeSI and FeSII clusters of active XDH-R135C differ from wild type, although broad similarities remain (Fig. 9A and Fig. 13). For the active XDH-R135C mutant, the temperature dependence of FeSI is shifted so that the signal is not as sharp at high temperatures, and that (unlike wild type) the two FeSI and FeSII signals cannot be readily separated by simply using different temperatures. The spin-Hamiltonian parameters were therefore evaluated by simulating the combined low temperature spectra. The FeSII signal of the active mutant is reasonably similar to that of wild type; on the other hand, the FeSI signal differs more significantly (Fig. 13). However, the signal remains axial, indicating that structural similarities probably remain. We conclude from this that the FeSI cluster in active XDH-R135C has an active site structure very similar to the wild type which is still able to distribute electrons within the protein. Unfortunately, it was impossible to obtain reproducible spectra from the inactive variant of XDH-R135C since the {alpha}{beta} dimeric form was rather unstable and the protein typically precipitated upon reduction.



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FIG. 13.
FeS EPR spectra of active XDH-R135C variant. a, experimental spectrum of FeSI and FeSII at 18 K; b, corresponding simulation made by adding equal double-integrated fractions of an FeSI simulation (c) and an FeSII simulation (d). c was calculated using gx,y,z = 1.928, 2.019; d was calculated with gx,y,z = 1.900, 1.995, 2.067. The arrow indicates the spectrum of a small quantity of Mo(V) present in this sample. A small quantity of flavin semiquinone (e) may also be present in the experimental spectrum.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Biosynthesis of the functional form of xanthine dehydrogenase is clearly a multistep process. In the case of the homodimeric mammalian XDH, this requires the incorporation of FAD, 2 Fe/S centers, and Moco into each subunit. It is not known whether this occurs during the synthesis of the protein or post-translationally before or after dimer formation. Formation of Moco itself is a complex process, involving binding of MPT and molybdenum and addition of the terminal sulfur ligand to molybdenum. It is well known that purified XO/XDH from diverse sources contains several inactive forms, including the desulfo and demolybdo variants and some lacking MPT. The need for multiple post-translational modifications poses a major hurdle for heterologous expression of the protein as well. The eukaryotic enzyme has been expressed previously in both Drosophila (27) and the baculovirus system (28, 29). However, these systems yielded preparations with no more than 10% functional enzyme. Even a recent report (30) on expression of Drosophila melanogaster XDH in Aspergillus nidulans showed that the recombinant enzyme is only 40% functional.

Biosynthesis of R. capsulatus XDH is even more complicated, requiring the assembly of an ({alpha}{beta})2 heterotetramer which is a dimer of dimers. Heterologous expression of the active enzyme in E. coli depends on whether E. coli has the ability to attach the terminal sulfur ligand, a process requiring a sulfurase enzyme. The data presented here show that the heterologous expression of R. capsulatus XDH in E. coli generates highly active enzyme, with 70% functionality compared with the enzyme purified after homologous expression in R. capsulatus. Besides, the E. coli expression system yields large quantities of active XDH in a reproducible manner, facilitating detailed structural and functional studies on the wild-type enzyme as well as mutants thereof.

The sulfuration of the molybdenum centers of the xanthine oxidase family of enzymes occurs by a mechanism that is not yet completely understood. In D. melanogaster, A. nidulans, and Arabidopsis thaliana, two-domain proteins designated Mal-1, HxB, and Aba3, respectively, have been implicated in the sulfuration of xanthine dehydrogenase and aldehyde oxidase (31, 32, 33). In bacteria, so far no protein with homologies to both protein domains has been identified. However, in order to be able to give rise to active sulfurated R. capsulatus XDH, E. coli must contain a protein capable of donating the terminal sulfur ligand to form active XDH. For the maturation of R. capsulatus XDH expressed in E. coli, XDHC was shown to be required for the sulfuration of Moco. XDH expressed heterologously in the absence of XDHC was shown to contain a desulfurated form of Moco.

Comparison of R. capsulatus XDH with bovine milk XO reveals an absorption spectrum similar in magnitude for both, but with differences in the overall shape of the visible range of the spectrum. The EPR data, showing that the FeSI center of R. capsulatus XDH is more symmetric than that of bovine XO, may in part explain the difference in the visible spectra of the two enzymes. Comparison of the substrate specificities of the two enzymes showed a similarity in the range of substrates oxidized. A notable difference between the two enzymes is that the R. capsulatus XDH is 5 times more active than fully active bovine milk XO. In addition, R. capsulatus XDH, like chicken XDH, is a true dehydrogenase that is not converted to the oxidase form upon proteolytic cleavage or oxidation of specific cysteine residues. R. capsulatus XDH, like bovine XO, oxidizes hypoxanthine to xanthine and xanthine to urate. Clearly, hypoxanthine and xanthine bind to the active site in different orientations, presenting C-2 and C-8 of the purine ring, respectively, to the molybdenum center. The tungsten-substituted XDH, while being catalytically inactive, should be able to bind the substrates at the active site. Crystallographic studies on the tungsten-substituted enzyme and its substrate complexes should provide insight into the diverse modes of substrate binding by the enzyme. EXAFS data of the R. capsulatus XDH alloxanthine-bound complex are very similar to that of bovine XO (26), showing a mono-oxo molybdenum site with the nitrogen atom of alloxanthine directly coordinated to molybdenum, and an Mo-S ligand in addition to the two thiolates from the MPT-dithiolene group. The recent crystallography of the XDH-alloxanthine complex suggested a similar coordination sphere so that the EXAFS and the crystallography data are in excellent agreement.

The higher turnover number of R. capsulatus XDH to oxidize its substrates can perhaps be explained by the fact that R. capsulatus utilizes purines as sole nitrogen source, degrading xanthine or hypoxanthine via urea completely to CO2 and ammonia, and is thus vital for rapid growth of the organism (6). In contrast, in humans the enzyme carries out the terminal reactions in excretory metabolism by oxidizing xanthine or hypoxanthine to uric acid, which is excreted in the urine. Loss of function of xanthine dehydrogenase in humans, a disease known as xanthinuria I (1), is often benign and may not even be diagnosed. However, because of the extreme insolubility of xanthine, some patients develop kidney malfunctions, with fatality in some extreme cases.

In the instance referred to here, the individual was hyperuricemic, and no XDH activity was detected in duodenal mucosa (4). However, Western blot analysis showed the presence of XDH protein. In view of this report it was surprising that expression of the R135C mutant of R. capsulatus XDH yielded a significant proportion of active enzyme in addition to the expected inactive form. In the mutant, the newly introduced Cys-135 in place of an arginine is located between Cys-134 and Cys-136 both of which serve as ligands to the FeSI complex. The UV-visible absorption spectra and EPR spectroscopy showed that FeSI is likely to be missing in the inactive mutant form but that the active mutant contains an apparently normal, although with a slightly different FeSI center.

These apparent contradictions can be rationalized by the hypothesis that mutationally derived Cys-135 forms a disulfide bond with either Cys-134 or Cys-136, leading to a failure in the assembly of the FeSI center. In the human tissues, the disulfide bond formation is virtually 100% complete because of the slow rate of synthesis of the XDH protein. However, in the case of the overexpressed recombinant R. capsulatus XDH, the rate of disulfide bond formation is unable to keep pace with the rate of synthesis of the protein, such that the Cys-134 and Cys-136 are able to participate in FeSI cluster formation in about 50% of the XDH molecules. It is also possible that the highly reducing cytoplasm of E. coli contributes to the attenuated rate of disulfide bond formation.

In the wild-type protein the guanidinium group of Arg-135 points away from FeSI to a basic cavity positioned within the protein. The amino acid side chain is about 6 Å away from one iron of FeSI and about 12 Å from the closest iron in FeSII. Modification of this amino acid would be expected to distort the protein fold in the vicinity of FeSI and possibly also in the vicinity of FeSII. We consider that the most likely cause of the spectroscopic differences is a conformational change in the peptide around the FeS clusters. It is also to be noted that the purified inactive mutant protein is a heterodimer, whereas the active mutant is a heterotetramer like unmutated XDH. These findings suggest that the absence of a functional FeSI center leads to altered conformation of the C-terminal Moco domain, such that it can neither incorporate Moco nor generate the dimer interface required for the formation of the heterotetramer.

In the future, the established expression system for R. capsulatus XDH in E. coli will serve for the generation of further site-specific mutants identified in xanthinuria I patients suffering from hyperuricemia and hyperuricosuria. Comprehensive biochemical, enzymatic, and molecular analyses of purified protein variants will help to define the specific underlying defects caused by mutations in XDH leading to xanthinuria I.


    FOOTNOTES
 
We dedicate this work to the memory of Professor Werner Klipp.

* This work was supported in part by Grant LE1171/2-1 from the Deutsche Forschungsgemeinschaft (to S. L.) and by Grant GM44283 from the National Institutes of Health (to K. V. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ";advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 49-531-391-5885; Fax: 49-531-391-8208; E-mail: S.Leimkuehler{at}tu-bs.de.

1 The abbreviations used are: XDH, xanthine dehydrogenase; XO, xanthine oxidase; MPT, molybdopterin; Moco, molybdenum cofactor; HPLC, high performance liquid chromatography; Ni-NTA, nickel-nitrilotriacetic acid; EXAFS, extended X-ray absorption fine structure; EPR, electron paramagnetic resonance; T, Tesla. Back

2 The EXAFSPAK suite of computer programs are available on-line at ssrl.slac.stanford.edu/exafspak.html. Back

3 G. N. George, I. J. Pickering, R. C. Prince, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Ralph Wiley (Duke University) for assistance in enzyme purification. We also thank Drs. H. Harris and I. J. Pickering for assistance with XAS data acquisition and Dr. S. J. George for recording some of the EPR spectra. Portions of this work were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Laboratory Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program.



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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