(Received for publication, November 14, 1994)
From the
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-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.
Xanthine dehydrogenase (XDH, ()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
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 activity, whereas xanthine oxidase (XO) by high xanthine/O
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
-terminal amino acid sequences of each fragment, the
20-kDa fragment is assigned to the NH
-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.
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.
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.
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.
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-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. ()
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D13221[GenBank].