©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Structure of Chicken Liver Xanthine Dehydrogenase
cDNA CLONING AND THE DOMAIN STRUCTURE (*)

(Received for publication, November 14, 1994)

Akira Sato (§) Tomoko Nishino (1) Kumi Noda (1) Yoshihiro Amaya (1) Takeshi Nishino (¶)

From the Department of Biochemistry, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, and theDepartment of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The amino acid sequence of chicken liver xanthine dehydrogenase (EC 1.1.1.204) was determined by cDNA cloning and partial amino acid sequencing of the purified enzyme. The enzyme consisted of 1358 amino acids with calculated molecular mass of 149,633 Da. In order to compare the structure of the chicken and rat enzymes, limited proteolysis was performed with the purified chicken liver xanthine dehydrogenase. When the enzyme was digested with subtilisin, it was not converted from the NAD-dependent dehydrogenase type to the O(2)-dependent oxidase type, in contrast with the mammalian enzyme. However, the enzyme was cleaved mainly into three fragments in a manner similar to that for the rat enzyme at pH 8.2 (20, 37, and 84 kDa) and retaining a full complement of redox centers. The cleavage sites were identified by determination of amino-terminal sequences of the produced fragments. It was concluded that the 20-kDa fragment was amino-terminal, the 84-kDa fragment carboxyl-terminal, and the 37-kDa fragment an intermediate portion in the enzyme protein. On the other hand, when the enzyme was digested with the same protease at pH 10.5, the sample contained only the 20- and 84-kDa portions and lacked the 37-kDa portion. The resultant sample possessed xanthine dichlorophenol indophenol reductase activity, indicating that the molybdenum center remained intact. The absorption spectrum showed the sample was very similar to deflavo-enzyme. From these results and sequence analyses, the domain structure of the enzyme is discussed.


INTRODUCTION

Xanthine dehydrogenase (XDH, (^1)EC 1.1.1.204) is a complex molybdo-flavoprotein that catalyzes oxidation of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of NAD or molecular oxygen. XDHs from various sources are proteins of M(r) 300,000 and are composed of two identical and independent subunits; each subunit contains one molybdopterin, two non-identical Fe2S2 centers, and a flavin adenine dinucleotide (Bray, 1975; Hille and Massey, 1985). It has been shown that the molecular weight of each subunit of the native enzyme prepared without proteolysis is 150,000 (Waud and Rajagopalan, 1986; Amaya et al., 1990). The full amino acid sequences of the enzymes have been determined from cDNA cloning from mammals (Amaya et al., 1990; Terao et al., 1991; Ichida et al., 1993; Wright et al., 1993) and insects (Keith et al., 1987; Lee et al., 1987; Rocher-Chambonnet et al., 1987; Houde et al., 1989).

The mammalian enzyme exists originally as a dehydrogenase, but it converts to an oxidase during extraction or purification procedures (Della Corte and Stirpe, 1968, 1972). XDH is characterized by high xanthine/NAD activity and low xanthine/O(2) activity, whereas xanthine oxidase (XO) by high xanthine/O(2) activity and negligible xanthine/NAD activity (Bray, 1975). Even the dehydrogenase form has a weak oxidase activity, but the oxidase activity of the mammalian enzyme is dramatically increased by protein modification in concomitant loss of the activity with NAD as a substrate (Stirpe and Della Corte, 1969; Waud and Rajagopalan, 1976; Nakamura and Yamazaki, 1982; Saito and Nishino, 1989; Hunt and Massey, 1992). This conversion occurs either by proteolytic cleavage or sulfhydryl oxidation of the protein molecule. By limited proteolysis of the mammalian enzyme with trypsin, the enzyme is converted to an XO type with concomitant cleavage into three fragments (20, 40, and 85 kDa) (Amaya et al., 1990). By determination of NH(2)-terminal amino acid sequences of each fragment, the 20-kDa fragment is assigned to the NH(2)-terminal portion, the 85-kDa fragment to the COOH-terminal portion and the 40-kDa fragment to an intermediate portion (Amaya et al. 1990). Although the three dimensional structure analysis by x-ray crystallography is still in progress (Eger et al., 1994), it was shown by electron microscopy that one subunit of milk XO consists of three submasses (Coughlan et al., 1986), suggesting that it is made up of three domains. Upon modification of the protein molecule, significant conformational changes seem to occur, particularly around the flavin (Massey et al., 1989; Saito et al., 1989), resulting in changes in reactivity of the flavin as well as loss of the NAD binding site (Nakamura and Yamazaki, 1982; Nishino and Nishino, 1989; Saito and Nishino, 1989; Hunt and Massey, 1992). From the sequence comparison (Amaya et al., 1990) and chemical modification studies (Nishino and Nishino, 1989), the redox centers are suggested to be located in three domains, e.g. the two iron-sulfur centers are in the 20-kDa domain, the FAD is in the 40-kDa domain, and the molybdopterin is in the 85-kDa domain (Amaya et al., 1990).

In contrast to the mammalian enzyme, the chicken enzyme has never been known to convert to an oxidase form. One of our long time goals is to determine why the chicken enzyme does not easily convert to an oxidase form. In this paper in order to obtain structural information on chicken XDH, we determined the amino acid sequence and identified the cleavage site of the enzyme protein by subtilisin. The domain structure of the enzyme is discussed in light of these findings.


EXPERIMENTAL PROCEDURES

Materials

Reverse transcriptase was obtained from SuperScript RNase H, Life Technologies, Inc. Restriction and modification enzymes were obtained from Takara Shuzo Co., Ltd. (Kyoto, Japan) or New England Biolabs, Inc. (Beverly, MA). Oligonucleotide primers were synthesized using a DNA synthesizer (model 381A, Applied Biosystems). XDH was purified from the livers of chickens fed on a high protein diet, as described previously by Nishino(1974). beta-NAD was purchased from Oriental Yeast Co.; subtilisin (protease type VIII), xanthine, and cytochrome c (bovine heart, type 3) were from Sigma; 2,6-dichlorophenol indophenol (DCPIP) was from WAKO (Japan). All other chemicals were of reagent grade and were used without additional purification.

Assays

Enzyme assays were carried out at 25 °C in 50 mM potassium phosphate buffer (pH 7.8) containing 0.4 mM EDTA in a final volume of 3.0 ml. The oxidation of xanthine (150 µM) was monitored by using the following acceptors and wavelengths: DCPIP (50 µM; 600 nm), O(2) (air-saturated buffer; 295 nm), and NAD (500 µM; 340 nm). The oxidation of NADH (100 µM) by DCPIP was followed at 600 nm. Enzyme concentration was determined from the value of 36.5 mM cm (Rajagopalan and Handler, 1967).

Isolation of cDNA Clones for Chicken XDH

The three peptide sequences of chicken XDH (Primers I-III, double underlined in Fig. 2) were used for design of primers for reverse transcriptase PCR. All PCR primers were 26-mer including HindIII (m-strand) or EcoRI (c-strand) sites at the 5`-end of the primers to facilitate subcloning. The nucleotide sequences of the primers were as follows: primer-Im (m-strand), CAAAGCTTTYTTYGTNAAY-GGNAARA; primer-IIm (m-strand), CAAAGCTTTNATGTGGAT(ACT)CARCCNA; primer-IIc (c-strand), CGAATTCNGGYTG(AGT)ATCCACATNAC; primer-IIIc (c-strand), TCGAATTCR-TANCCNGTRAARAAYTT (where N is any nucleotide, Y is pyrimidine, and R is purine).




Figure 2: Nucleotide sequence of chicken XDH cDNA and its deduced amino acid sequence. Nucleotide sequence is connection of following clones: CXDH1(-129-1594), CXDH4(1595-2162), CXDH2(2163-3488), and CXDH7(3489-4202). The peptide sequences (total 683 amino acid residues) determined by Edman degradation are underlined with solid lines.



Poly(A) RNA was obtained by the method of Chomczynski and Sacchi(1987) from a liver of chicken fed on a high protein diet. Reverse transcriptase PCR reactions were performed essentially according to the method of Compton(1990). The first cycle PCR reactions were performed under low stringency annealing conditions (annealing at 40 °C for 2 min, extension at 72 °C for 3 min, and denaturation at 94 °C for 1 min; 10 cycles) and then high stringency annealing conditions (annealing at 50 °C; 20 cycles). Approximately 700 and 500 base pair fragments were obtained from the first round PCR using primer-sets, Im-IIc and IIm-IIIc, respectively. These PCR products were further amplified by a second cycle PCR reaction (annealing at 50 °C, 30 cycles). Both of the fragments were inserted into pBluescript SK-, and then subjected to sequence analysis. It was found that the deduced amino acid sequence of the PCR products (fragment 1, 762 base pairs; fragment 2, 414 base pairs) contained peptide sequences completely identical to the those known from the purified enzyme. Because fragment 2 contained an internal HindIII site (at 66 base pairs from the 5`-end), it was cleaved into a shorter fragment during subcloning. A chicken liver cDNA library (CL1002b: Clontech Laboratory, Inc.) was screened by plaque hybridization initially using the fragments of the PCR products as probes. In initial screening, four positive clones (CXDH1-CXDH4, Fig. 1) were isolated. The EcoRI 450-base pair or 3`-end 145-base pair fragment of the CXDH2 insert was used for rescreening of the library, and three positive clones were isolated (CXDH5-CXDH7, Fig. 1). Inserts of the positive clones from cDNA library were subcloned and analyzed. All the methods of cDNA cloning and sequence analyses were performed essentially according to the method of Maniatis et al. (1982).


Figure 1: Restriction map of chicken XDH cDNA. The upperpart of the figure shows restriction sites of chicken XDH cDNA. The sequence corresponding to the coding region is indicated by the closedbox, and 5`- and 3`-noncoding regions of the cDNA are indicated by openboxes. PCR products used for probes in the initial screening stage are indicated by dottedboxes. Inserts of the cDNA clones are shown in the lowerpart. The abbreviations of restriction enzymes are: H, HindIII; S, SacI; E, EcoRI. Kbp, kilobase pairs.



Nucleotide Sequence Analysis

The nucleotide sequencing of the double-strand plasmids was determined by the dideoxynucleotide chain termination method using synthetic oligonucleotide primers complementary to the vector or the XDH cDNAs (Sanger et al., 1977; Hattori and Sakaki, 1986).

Digestion of Chicken Liver XDH with Subtilisin and NH(2)-terminal Sequences of Cleaved Peptides

Subtilisin treatment was carried out at 30 °C in buffer mixture, pH 8.2 (a mixture of identical volumes of 0.1 M pyrophosphate buffer, pH 8.5, and 0.05 M potassium phosphate buffer, pH 7.8) for 40 h or carried out in 0.2 M Na(2)HPO(4)-NaOH pH 10.5 for 20 h. The weight ratio of subtilisin to xanthine dehydrogenase was 1 to 10. During incubation, aliquots of nicked XDH were withdrawn at various incubation time followed by addition of phenylmethanesulfonyl fluoride (2 mM final) and were subjected to SDS-PAGE. SDS-PAGE was performed on 10% gel according to the method of Laemmli(1970). In order to determine the NH(2)-terminal sequence of the fragments, the samples of SDS-PAGE were electroblotted to Problott membrane (polyvinylidene difluoride membrane, Applied Biosystems) and the blotted membrane was cut into small pieces and subjected to amino acid sequence analysis.

Partial Amino Acid Sequence Analysis of XDH

For amino acid sequence analysis of internal peptide fragments, the purified enzyme was digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Boehringer Mannheim), lysyl endopeptidase (Boehringer Mannheim), V8 protease (ICN Biochemicals), or cyanogen bromide. The peptides were separated with two steps of high performance liquid chromatography using µBondapak (Waters) and Capcellpak (Shiseido, Tokyo) columns. Appropriate peptides, each of which was eluted as a single sharp peak, were subjected to amino acid sequence analysis.

Protein Sequence Analysis

Amino acid sequence analysis was performed by the automated Edman degradation method with a gas phase sequencer (model 477A), equipped with a phenylthiohydantoin-derivative analyzer (model 120A, Applied Biosystems).


RESULTS AND DISCUSSION

Molecular Cloning of cDNAs for Chicken Liver Xanthine Dehydrogenase and Deduced Amino Acid Sequence

cDNA clones for chicken liver XDH were isolated as described under ``Experimental Procedures.'' The nucleotide sequence and the predicted amino acid sequence are shown in Fig. 2. The clones covered along 4325 bases of chicken XDH mRNA. Sequence analyses revealed an open reading frame of 4074 base pairs encoding 1358 amino acid residues with calculated molecular weight of 149,633. The deduced amino acid sequence from the cDNA clones was completely identical to the partial peptide sequence known from the purified chicken enzyme (683 amino acid residues, Fig. 2, underlined).

Identification of the Cleavage Sites of Peptide Fragments after Limited Proteolysis of Purified Chicken Liver XDH with Subtilisin

In order to obtain structural information, SDS-PAGE analysis was performed with the purified enzyme after incubation at pH 8.2 with subtilisin as described under ``Experimental Procedures'' and shown in Fig. 3. Under the conditions used, the NAD-dependent activities of chicken liver xanthine dehydrogenase were found to be inactivated almost completely after 40 h of digestion. On the other hand, xanthine-oxygen reductase activity was slightly decreased, whereas xanthine-DCPIP activity was maintained almost at the same level of its initial activity (data not shown). The results differ from those with the rat enzyme in that no increase of xanthine-oxygen reductase activity was observed with chicken liver XDH on digestion with protease. As this report concentrates mainly in the structural aspects, the detailed functional analysis of the nicked enzyme digested at pH 8.2 will be described elsewhere. The fragmentation pattern is shown in Fig. 3and is very similar to that reported by Coughlan et al.(1979). After 1 h of digestion, three major fragments having molecular masses of 84 kDa (designated as fragment 1), 37 kDa (fragment 3), and 20 kDa (fragment 6) (estimated from SDS-gel electrophoresis) were observed, but after 3 h of digestion, 59-kDa (fragment 2) and 24-kDa (fragment 5) fragments had become apparent and increased with time until 40 h. After 5 h of digestion, the 37-kDa fragment had disappeared almost completely, but the 32-kDa fragment (fragment 4), which existed as a minor band at 1 h of digestion, became significant. The 84-kDa fragment was apparently decreased after 3 h, but still existed even after 40 h of incubation. The two electrophoresis gel samples, after 1- and 24-h digestions, were electroblotted to Problott membrane and each blotted peptide was analyzed by the Edman degradation method as shown in Table 1. The NH(2)-terminal amino acid residues of the 20-kDa fragment correspond to the sequence Ala^2-Lys^18 of the cDNA deduced sequence. Although the apparent molecular mass of the 20-kDa fragment decreased to about 18 kDa after 24 h of digestion (Fig. 3), it was found that the NH(2)-terminal amino acid sequence was identical, indicating that this fragment was degraded into a smaller fragment at its COOH terminus. The NH(2)-terminal sequences of the 37- and 32-kDa fragments correspond to Glu-Leu and Glu-Trp, respectively. The NH(2)-terminal sequence of the 84-kDa fragment correspond to Leu-Val. The NH(2)-terminal sequence of 20 amino acids of the 24-kDa fragment correspond to Asn-Leu (Fig. 2, underlined). Therefore, it was concluded that the enzyme was at first cleaved into three fragments in similar way as the rat enzyme, these fragments were considered to be further degraded at the COOH termini into smaller fragments by prolonged incubation. The 32-kDa fragment was the degraded product of the 37-kDa fragment, and the 59- and 24-kDa fragments were from the 84-kDa fragment. It should be noted that the calculated molecular masses of the putative peptide fragments from Glu to Gln (38,943 Da), from Leu to Ser (59,288 Da) and from Asn to Ala (24,612 Da) agreed approximately with the molecular masses of 37-, 59-, and 24-kDa bands, respectively, which were estimated from gel electrophoresis. However, the calculated molecular mass of the peptide from Met^1 to Lys (26,843 Da) is significantly larger than those estimated from SDS-PAGE, suggesting that the COOH terminus of this fragment was already degraded even after 1 h of digestion. A scheme of the fragmentation is illustrated in Fig. 4.


Figure 3: Time course of xanthine dehydrogenase fragmentation by subtilisin. Xanthine dehydrogenase was treated with subtilisin as described under ``Experimental Procedures'' using the buffer mixture (pH 8.2) at 30 °C for 0 (lane 1), 1 (lane2), 3 (lane3), 5 (lane4), 7 (lane5), 14 (lane6), 20 (lane7), 24 (lane8), and 40 (lane 9) h. Fragment numbers are designated at left, while molecular masses of the marker sample (lane10) are indicated at right.






Figure 4: Time-dependent fragmentation in chicken liver xanthine dehydrogenase with subtilisin. The diagram is based on the data of Table 1, and the sizes of peptide fragments are derived from the data of Fig. 2. 1, native enzyme; 2, after 1 h of digestion; 3, after 24 h of digestion.



Isolation of the Complex of Iron-Sulfur Centers and the Molybdenum-containing Domains

Coughlan et al.(1979) reported the isolation of the molybdenum domain of chicken liver xanthine dehydrogenase, which contained both the molybdenum and two iron-sulfur centers, and suggested that the molybdenum and the iron-sulfur centers might be located in the same domain having a mass of 65 kDa. However, this proposal is not consistent with prediction from the analysis of the protein sequence; the iron-sulfur centers are located in the NH(2)-terminal portion whereas the molybdenum is located in the COOH-terminal portion. In order to explain the apparent discrepancies between the sequence prediction and the results of proteolysis, we repeated the experiment similar to that of Coughlan et al.(1979). After digestion of purified XDH under different conditions (pH 8.2 or 10.5), the samples were subjected to the gel-filtration column. The elution patterns of gel filtration chromatography of each sample are illustrated in Fig. 5a. The elution position and the height of the first and the second peaks of absorbance at 280 nm were not identical in the two samples, suggesting the formation of different products. The product digested at pH 8.2 had a major peak at the higher molecular weight position and a minor peak at lower molecular weight, which contained subtilisin and further degraded small fragments. On the other hand, the product digested at pH 10.2 had a smaller first peak with slightly lower M(r) than the sample digested at pH 8.2 and a larger second peak, indicating that majority of the enzyme was digested into small fragments under alkaline conditions. The first peaks of the samples, digested either at pH 8.2 or at pH 10.5, possessed xanthine-DCPIP reductase activity and were therefore expected to contain intact molybdenum centers. These were collected and subjected to the SDS-PAGE analysis. As shown in Fig. 5b, the products digested at pH 8.5 contained all the fragments as described in the previous section. However, the product digested at pH 10.5 contained 20-, 24-, 59-, and 84-kDa fragments, but lacks a 37- or 32-kDa fragment. This suggests that the 37-kDa fragment, which is the intermediate portion of the whole sequence, has been removed from the complex and digested further into smaller fragments by digestion with subtilisin under alkaline conditions. It was concluded that the products digested at pH 10.5 contained a complex of the NH(2)-terminal fragment (20 kDa) and the COOH-terminal fragment (84 kDa or 59 plus 24 kDa) of the enzyme. These fragments might be tightly associated with each other and be dissociated under denaturation conditions.


Figure 5: Panel a, gel filtration chromatography using Ultrogel AcA22 of subtilisin-treated xanthine dehydrogenase. Partially digested protein sample was subjected on Ultrogel AcA22 by irrigation with 50 mM potassium phosphate buffer, pH 7.8, containing 0.4 mM EDTA. Part A, the sample incubated with subtilisin for 40 h at pH 8.2; part B, the sample incubated with subtilisin for 20 h at pH 10.5. One unit of DCPIP reductase activity was defined as 1 absorbance change at 600 nm/min. Panel b, the eluted samples from gel filtration (a). Fractions 35-41 (the sample incubated at pH 8.2) or fractions 37-43 (the sample incubated at pH 10.5) were pooled, concentrated, and subjected to SDS-PAGE. Lane 1, sample digested at pH 10.5; lane 2, sample digested at pH 8.2; lane 3, molecular markers of 94, 67, 43, 30, and 20.1 kDa. Panel c, absorption spectra of the eluted samples from gel filtration: 1, native XDH (-); 2, the sample incubated at pH 8.2(- - -); 3, the sample incubated at pH 10.5(- -); 4. the difference spectrum between the native samples and samples incubated at pH 10.5 ( . . . ).



The absorption spectrum and the difference spectrum between the native and the digested product indicate that the sample digested at pH 10.5 is very similar to that of the deflavo-enzyme (Nishino et al., 1989), suggesting that it contains the molybdenum and iron-sulfur centers (Fig. 5c). It should be noted that the sample possesses xanthine-DCPIP activity and therefore the xanthine binding site is intact, consistent with the sample containing the molybdenum center. On the other hand, the absorption spectrum of the sample digested at pH 8.2 is not significantly different from that of normal enzyme, indicating that it contains its full complement of the redox centers.

Functional Domains of Chicken XDH

The amino acid sequence of the chicken liver enzyme is compared to that of the rat enzyme (Amaya et al., 1990), as shown in Fig. 6. The predicted amino acid sequence of the chicken enzyme is homologous with those of the rat and Drosophila melanogaster enzymes along the entire sequences (70% and 53% identical without counting gaps, respectively). The chicken liver enzyme consists of 1358 amino acids, a little longer than the mammalian enzyme, mainly due to an insertion at the NH(2)-terminal fragment portion. The human enzyme consists of 1334 (Ichida et al., 1993) and the rat enzyme 1331 amino acids. During the course of this work, we resequenced the cDNA, as well as the purified enzyme protein, of the rat enzyme and found that the previously reported sequences (Amaya et al., 1990) had an error of deletion of 12 amino acids (shown in Fig. 6). The revised sequence of the rat liver XDH was completely identical to that of the rat macrophage enzyme reported recently by Chow et al.(1994), indicating no difference between rat liver and macrophage XDH mRNAs. The amino acid sequence of the chicken enzyme is essentially similar to that of the rat enzyme. The lysine residues, which have been attacked by tryptic digestion and are considered to be responsible for dehydrogenase to oxidase conversion (Amaya et al., 1990) are also conserved. Among 3 cysteine residues, corresponding to Cys, Cys, and Cys of the rat enzyme, which were modified by fluorodinitrobenzene and are expected to be candidates for the residue(s) responsible for D/O conversion, (^2)1 of them (Cys) was conserved, but 2 of them (Cys and Cys) were replaced by arginine and phenylalanine, respectively. Probably such replacements might explain why the chicken liver enzyme is not converted to the oxidase form by sulfhydryl oxidants.


Figure 6: Comparison of the amino acid sequence of the three fragments of chicken XDH with those of the rat enzyme. Rat enzyme fragments were formed by tryptic digestion, while chicken enzyme was by subtilisin. The amino acid sequence of chicken XDH is from this study (Fig. 2); that of rat is from Amaya et al.(1990) with revision of 12 amino acid residues (boxed) insertion between Leu and Gln of the previously reported sequence. Gaps are introduced to increase the similarity, and matching amino acids among chicken and rat are denoted by dottedboxes. Conserved cysteine residues between chicken and rat enzymes are dotted on the top. Amino acid residues labeled with FSBA (Nishino and Nishino, 1989) or fluorodinitrobenzene are denoted by openboxes.



Peptide fragments of 84, 37, and 20 kDa were generated by digestion at pH 8.2 with subtilisin as described above. The positions of the chicken enzyme nicked by proteolysis are not very far from those of the rat enzyme obtained by trypsin digestion. As described in the previous section, the 20-kDa fragment is assigned to the NH(2)-terminal portion, the 84-kDa fragment to the COOH-terminal portion, and the 37-kDa fragment to intermediate portions, essentially similar to the rat enzyme. As was suggested by electron microscopy the subunit of milk XO is made up of three submasses (Coughlan et al., 1986), the striking similarity in limited proteolysis and in amino acid sequence among xanthine oxidizing enzymes suggests that the chicken liver enzyme might also be consist a three-domain structure. The model of the domain structure is illustrated in Fig. 7. As is the case of the rat enzyme, these domains seem to associate strongly with each other and to dissociate under denaturation conditions. However, as described in the previous section, the intermediate 37-kDa domain seems to be easily removed and be digested under alkaline conditions.


Figure 7: The proposed domain structure model of a single subunit of chicken liver xanthine dehydrogenase. The enzyme subunit is likely to consist of three domains having masses of 20, 37, and 84 kDa containing the iron-sulfur centers, the FAD, and the molybdenum centers, respectively. When the enzyme is digested at pH 8.2, two interconnecting segments between three domains might be digested resulting in a nicked enzyme. On the other hand, when the enzyme is digested at pH 10.5, the FAD-containing domain might be removed and result in a complex of 20- and 84-kDa domains.



The 20-kDa fragment portion is well conserved among chicken, rat, and D. melanogaster enzymes. This portion contains conserved cysteine cluster residues, 8 of which are located in a hydrophobic environment as described previously (Amaya et al., 1990). In the rat enzyme, these residues are assumed be associated with the 2Fe/2S-type redox centers. Cys-Cys of the chicken enzyme is consistent with the consensus sequence pattern of ferredoxin type iron-sulfur centers in the data base of protein sequence patterns (Bairoch, 1993). There is no cysteine cluster other than in this domain; therefore, it is unlikely that the iron-sulfur centers exist other than this domain. This is also compatible with the result that the product digested at pH 10.5 contained 20-kDa fragment as well as the iron-sulfur centers. It has been pointed out that there is a nucleotide binding site motif (G-X-G-X-X-G) in this cysteine cluster, and therefore it is considered to be the NAD binding site (Wright et al., 1993). However, it seems unlikely that the NAD binding site and the iron-sulfur centers share the same amino acid residues.

The 84-kDa fragment portions are moderately conserved between the chicken and rat enzymes. A putative molybdenum binding site was suggested by sequence analysis among XDH and other molybdo-enzymes (Wooton et al., 1991). This sequence is also conserved in chicken XDH; however, it is not well conserved among all the reported XDH sequences. There is no direct evidence that this domain contains the molybdenum center, but the chemical modification sites of the rat enzyme by fluorodinitrobenzene, which affect the rate of release of urate (Nishino et al., 1982) and therefore are considered to be located near the molybdenum center, have been identified to be Lys754 and Lys771 which exist in the 85-kDa domain. (^3)

Compared with the 20- and 84-kDa fragment portion, the 37-kDa fragment portion (residues 249-596 of chicken enzyme) is not well conserved. This fragment portion was assumed to be the NAD-associating domain from affinity-label experiments of chicken liver XDH with 5`-p-fluorosulfonylbenzoyladenosine (FSBA) (Nishino and Nishino, 1989). Although it is not clear at the moment whether the residue that reacts with FSBA actually contributes to the nucleotide binding, this residue should be located near the NAD binding site because FSBA exhibits many of the characteristics of an active site-directed reagent for the NAD binding site (Nishino and Nishino, 1987). The FSBA-modified amino acid residue is identified to be Tyr of the chicken enzyme and is well conserved among all the sequences from various sources including mammals and insects, except in the sequence of the human enzyme reported by Wright et al.(1993). The sequence reported by Wright et al. has weaker homology with those of other mammalian enzymes (49.4% identity with the rat enzyme) and is strikingly different from the sequence of the human enzyme reported by Ichida et al.(1993), which has 90.2% identity with the rat enzyme. It is possible that the sequence reported by Wright et al. might be the sequence of another related enzyme of the family of molybdenum containing iron-sulfur flavoproteins such as aldehyde oxidase rather than xanthine dehydrogenase itself. However, the reason for this discrepancy is not clear at present and must await characterization of the enzyme expressed from the cDNA isolated by Wright et al.. The results that the complex of 20- and 84-kDa fragment, the product of digestion at pH 10.2 (Fig. 7), contains both the iron-sulfur and the molybdenum centers but lacks the FAD centers, suggests that the 37-kDa fragment is the FAD associating domain. As NAD reacts with FAD (Schopfer et al., 1989), it is reasonable that the NAD-associated domain is also the FAD associating domain. However, as the typical known motif of nucleotide binding sites has not been found in this domain, it is possible that the nucleotides are not associated only to a single domain. Precise location of these redox center must await x-ray crystallographic analysis, which is now in progress (Eger et al., 1994). The relatively low homology in amino acid sequence of the intermediate domain suggests that the protein conformation is different in this portion between the rat and chicken enzymes. This will be discussed in more detail elsewhere with functional analysis of the nicked enzyme why the oxidase activity does not increase in chicken liver XDH.


FOOTNOTES

*
This work was supported by Grants-in-aid 04225229 and 05670152 for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a research grant for intractable disease from the Japanese Ministry of Health and Welfare. Part of this work has been presented in a preliminary form (Nishino, T., Nishino, T., Sato, A., Page, T., and Amaya, Y.(1993) Proceedings of 11th International Symposium on Flavins and Flavoproteins, July 27-31, 1993, Nagoya, Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D13221[GenBank].

§
Present address: Dept. of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 724, Japan.

To whom correspondence should be addressed. Tel.: 81-3-3822-2131 (ext. 422); Fax: 81-3-5685-3054.

(^1)
The abbreviations used are: XDH, xanthine dehydrogenase; XO, xanthine oxidase; DCPIP, 2,6-dichlorophenol indophenol; FSBA, 5`-p-fluorosulfonylbenzoyladenosine; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

(^2)
T. Nishino, and T. Nishino, manuscript in preparation.

(^3)
T. Nishino and T. Nishino, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Vincent Massey, the University of Michigan, for helpful discussions and critical reading of the manuscript.


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