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Volume 270, Number 8, Issue of February 24, 1995 pp. 3534-3550
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Molecular Characterization of hxA, the Gene Coding for the Xanthine Dehydrogenase (Purine Hydroxylase I) of Aspergillus nidulans(*)

(Received for publication, August 1, 1994; and in revised form, November 16, 1994)

Annie Glatigny ,  Claudio Scazzocchio (§)

From the Institut de Génétique et Microbiologie, Unité Associée au CNRS 1354, Université Paris-Sud, Bâtiment 409, Centre d'Orsay, F-91405 Orsay, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have cloned and sequenced the hxA gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans. The gene codes for a polypeptide of 1363 amino acids. The sequencing of a nonsense mutation, hxA5, proves formally that the clones isolated correspond to the hxA gene. The gene sequence is interrupted by three introns. Similarity searches reveal two iron-sulfur centers and a NAD/FAD-binding domain and have enabled a consensus sequence to be determined for the molybdenum cofactor-binding domain. The A. nidulans sequence is a useful outclass for the other known sequences, which are all from metazoans. In particular, it gives added significance to the missense mutations sequenced in Drosophila melanogaster and leads to the conclusion that while one of the recently sequenced human genes codes for a xanthine dehydrogenase, the other one must code for a different molybdenum-containing hydroxylase, possibly an aldehyde oxidase. The transcription of the hxA gene is induced by the uric acid analogue 2-thiouric acid and repressed by ammonium. Induction necessitates the product of the uaY regulatory gene.

INTRODUCTION

Xanthine dehydrogenases are ubiquitous enzymes that have been thoroughly studied from the biochemical point of view. Their structure is conserved throughout evolution. They are dimers, with each monomer of 150 kDa containing a complex electron transport chain with a pterin-bound molybdenum, a flavin, and two iron-sulfur centers as cofactors. NAD is usually the terminal electron acceptor (Coughlan, 1980; Rajagopalan, 1991; Wootton et al., 1991). A number of enzymes related to the xanthine dehydrogenases have been described. These show the same overall general structure, but different substrate specificities. Among these are the aldehyde oxidases from a wide range of metazoans, including mammals and Drosophila melanogaster, and the pyridoxal dehydrogenase from D. melanogaster (Krenitsky et al., 1972, 1974; Branzoli and Massey, 1974; Courtright, 1976).

The M(r) of the Aspergillus nidulans xanthine dehydrogenase has been estimated to be 304,000. Its cofactor content is identical to that of other enzymes of this group (Scazzocchio et al., 1973; Lewis et al., 1978; Scazzocchio and Sealy-Lewis, 1978; Mehra and Coughlan, 1989). Many loss-of-function mutations of the A. nidulans enzyme have been obtained (Darlington et al., 1965; Alderson and Scazzocchio, 1967; Darlington and Scazzocchio, 1967). Two classes of substrate specificity mutations have also been obtained. The first shows an altered kinetics of inhibition by allopurinol (Scazzocchio, 1966). The second shows a number of pleiotropic effects, the most striking of which is that while the wild-type enzyme hydroxylates 2-hydroxypurine at position 8, the mutant enzyme hydroxylates this same analogue at position 6 (Scazzocchio and Sealy-Lewis, 1978). All these mutations map in one gene, hxA, located in chromosome V. A fine structure map has been constructed, which positions all substrate specificity mutations in a discrete domain of the gene. (^1)Transformation techniques (see below) permit the physical location of any mutation and introduction of new ones at selected places in the gene. The enzyme of D. melanogaster has also been subject to a thorough genetic analysis (Chovnick et al., 1977, 1990; Gray et al., 1991; Hughes et al., 1992a, 1992b; Doyle and Bray, 1994). The selection techniques that are available for A. nidulans and D. melanogaster are not identical; thus, the classes of mutations extant in the two organisms are only partially overlapping. In particular, substrate specificity mutations have been obtained only in A. nidulans.

A second enzyme, purine hydroxylase II, has been described in A. nidulans. This is physiologically a nicotinate hydroxylase, and it is inducible by 6-hydroxynicotinate (Sealy-Lewis et al., 1979) and controlled with other enzymes of the nicotinate degradation pathway (Scazzocchio et al., 1973; Scazzocchio, 1980, 1994). The enzyme accepts hypoxanthine, but not xanthine, as a substrate and presents some interesting kinetic and mechanistic peculiarities (Lewis et al., 1978; Coughlan et al., 1984; Scazzocchio, 1994 (for review)). Other nicotinate hydroxylases have been described in bacteria, but none has a hypoxanthine hydroxylase activity (Hirschberg and Ensign, 1972; Imhoff and Andreesen, 1979). Thus, a comparison of the two purine hydroxylases of A. nidulans would be of evolutionary and mechanistic interest.

The genetic system of A. nidulans has provided a complete picture of the gene-protein relationships of the molybdenum-containing enzymes. The structural genes coding for the three molybdenum-containing enzymes, xanthine dehydrogenase, purine hydroxylase II, and nitrate reductase, have been identified and mapped, and many alleles in each have been characterized and ordered in fine structure maps (Scazzocchio et al., 1973; Scazzocchio and Sealy-Lewis, 1978; Scazzocchio, 1980; Tomsett and Cove, 1979). The genes involved in the molybdenum cofactor biosynthesis have been identified. The concept of a common molybdenum-containing cofactor was first derived from the existence of six genes in which mutations led to the loss of nitrate reductase and xanthine dehydrogenase activities (Pateman et al., 1964). Later it was found that purine hydroxylase II also requires the molybdenum cofactor (Scazzocchio, 1973; Scazzocchio, 1980). Another gene, hxB, codes for a protein needed for the substrate-specific hydroxylation activity of both purine hydroxylases I and II, but not for some ancillary activities such as the NADH dehydrogenase activity (Scazzocchio et al., 1973; Sealy-Lewis et al., 1978; Scazzocchio, 1980, 1994). Mutations in this gene do not affect nitrate reductase activity. This gene would seen to code for a protein necessary for a post-translational modification and may well be isofunctional with the ma-l gene of D. melanogaster, coding for a protein involved in the generation of the molybdenum-bound sulfur atom (Wahl et al., 1982).

The three molybdoproteins of A. nidulans are inducible, each by a specific metabolite. Each enzyme is coinduced with other enzymes of the same pathway. The specific regulatory genes involved in each induction process have been genetically characterized (Cove, 1979; Scazzocchio, 1994 (for reviews)) and, in the cases of the regulatory genes involved in nitrate and purine assimilation, cloned and sequenced (Burger et al., 1991a, 1991b; Suárez et al., 1991). (^3)The protein coded by the uaY gene mediates uric acid induction of at least eight activities of the purine utilization pathway, including xanthine dehydrogenase (Scazzocchio et al., 1982; Scazzocchio, 1994). The transcription of the genes of the purine utilization pathway necessitates a specific inducer, uric acid, but occurs efficiently only in the absence of ammonium or glutamine. However, neither the response to specific induction nor ammonia repression is identical in all genes of the pathway, with the basal noninduced level of xanthine dehydrogenase being considerably higher than that of the uric-acid permease (Gorfinkiel et al., 1993). A GATA factor, the product of the areA gene, mediates ammonia and glutamine repression of possibly all genes coding for enzymes and permeases involved in the utilization of nitrogen sources (Arst and Cove, 1973; Kudla et al., 1990).

The genomic and cDNA sequences of the genes coding for urate oxidase and the urate-xanthine permease have been reported. The transcription of these genes under different conditions and in wild-type and mutant backgrounds has been studied (Oestreicher and Scazzocchio, 1993; Gorfinkiel et al., 1993).

The cloning and sequencing of the hxA gene are of dual interest. From the point of view of regulation, it will contribute to our understanding of the different responses to uric acid induction and ammonium repression of different genes responding to the same regulatory gene products. From the point of view of enzymology, the comparison of enzymes from organisms as different as mammals, insects, and fungi will allow the identification of putative, discrete functional domains. In addition, the availability of a unique set of mutations will permit the definitive identification of functional domains and residues determining substrate binding and specificity.

EXPERIMENTAL PROCEDURES

Strains, Gene Libraries, and Transformation Techniques

The A. nidulans strains used in this work were pabaA1 (wild-type strain), CS2008 (wA4, pyroA4, biA1, argB2, hxA1, hxnR2), SL453 (pabaA1, pantoB100, hxA5), CS243 (yA2, pyroA4, riboC3, uaY6), NA2 (pabaA1, cbxC34, uaY205), and CS2227 (pabaA1, areA600). yA2 and wA4 result in yellow and white conidiospores, respectively; pabaA1, pantoB100, biA1, and argB2 indicate auxotrophies for p-aminobenzoic acid, D-pantothenic acid, biotin, and arginine, respectively; and cbxC34 results in resistance to carboxin (Scazzocchio et al., 1982). None of these makers has any effect on the regulation of the hxA gene. areA600 is an early chain termination mutation in the wide domain regulatory gene areA (Kudla et al., 1990). hxnR2 is a mutation in the regulatory gene of the nicotinate utilization pathway and results in noninducibility of purine hydroxylase II (nicotinate hydroxylase) (Scazzocchio, 1980). uaY6 is a null mutation resulting from an early frameshift in the uaY gene.^3uaY205 is a 16-base pair deletion resulting in a frameshift in the carboxyl terminus of the uaY open reading frame. (^4)The hxA alleles used in this work or mentioned in the text are hxA1, hxA5, hxA14, and hxA15, displaying complete loss of xanthine dehydrogenase activity (Darlington et al., 1965). hxA101 and hxA102 were selected by their ability to utilize 2-hydroxypurine as sole nitrogen source and altered specificity in the position of hydroxylation of this analogue from position 8 to 6 (Scazzocchio and Sealy-Lewis, 1978). hxA143 has been isolated as a mutation enabling the use of hypoxanthine as sole nitrogen source in the presence of allopurinol (Scazzocchio, 1966) (the conditions of selection were identical to those described by Scazzocchio et al.(1973)). All these mutations have been shown to map within the hxA gene.^1

Plasmids and Gene Libraries

Escherichia coli strain JM109 (Yanish-Perron et al., 1983) was used for routine plasmid preparation. A gene library of A. nidulans DNA partially restricted with Sau3A1 was constructed in plasmid pFB39, carrying a beta-lactamase gene and an intact A. nidulans argB gene (Buxton et al., 1989; Berse et al., 1983). A gene library constructed from A. nidulans DNA partially restricted with Sau3A1 in EMBL4 was kindly provided by C. M. Lazarus and J. Turner.

Transformation Techniques

Transformation of E. coli was performed according to Hanahan(1983). Transformation with whole DNA extracted from A. nidulans was carried out in E. coli strain DH1 (Bachmann, 1987). Transformation of A. nidulans was according to Tilburn et al. (1983). The mapping of the hxA5 mutation by transformation was as follows. A protoplast preparation of strain SL453 was divided in aliquots of 200 µl; these were incubated with 5 µg of each of the plasmids shown in Fig. 1, including plasmid pBAN884 as a positive control. This plasmid transforms all hxA alleles so far tested at frequencies of >100 transformants/µg. hxA transformants were selected on minimal medium containing hypoxanthine (100 µg/ml) as sole nitrogen source. We found that the only subclone that yielded hxA transformants was pBAN873X (pBAN876X, which contains the same sequence in opposite direction, was not tested). The hxA sequences of this plasmid were subcloned in Bluescript KS, using appropriate restriction sites internal to the plasmid, and the whole procedure was repeated with these subclones.

Figure 1: Restriction map of plasmid pBAN884 and sequencing strategy. The continuoussegment indicates the BglII fragment cloned in plasmid pBAN884. The shortarrows indicate the extent and direction of each sequencing reaction. The whole BglII-SacII region was sequenced on both strands; the direction of transcription is from left to right. This was determined by hybridization with the single-stranded plasmids pBAN873X and pBAN876X, which contain the same fragment cloned in opposite orientations. Plasmid pBAN816C was used as a probe for screening the ZAPII cDNA library. B, BglII; C, ClaI; S, SacII; Xb, XbaI. Other plasmids used either for sequencing or transformation experiments are also shown. kb, kilobase.


Media and Growth Conditions

The media and growth conditions for A. nidulans were as described by Scazzocchio et al.(1982). The mycelia for RNA extraction were grown for 20 h at 25 °C and shaken at 130 rpm in appropriately vitamin-supplemented minimal medium with 5 mM urea (noninducing, nonrepressing conditions) or 1.25 mM ammonium D(+)-tartrate (2.5 mM in ammonium ion, limiting ammonium, partially repressing conditions). Induction was carried out by adding 27 µM 2-thiouric acid after 15 h of growth. Repression was performed by adding 5 mM ammonium D(+)-tartrate (10 mM ammonium ion) together with the gratuitous inducer.

Isolation of Nucleic Acids

Preparation of DNA and RNA from A. nidulans was as described by Sherman et al. (1978) as modified by Nelson et al.(1989) and Lockington et al.(1985). Double-stranded plasmid were prepared as described by Birnboim and Doly(1979). Single-stranded DNAs from E. coli JM109 cells containing Bluescript phagemid were recovered according to the instruction manual from Stratagene (La Jolla, CA).

Sequencing

DNA sequences were determined using the dideoxynucleotide chain termination procedure (Sanger et al., 1977) on single- or double-stranded templates. All of the clones were sequenced on both strands using T7, SK, KS, T3, or specific primers (Stratagene). For sequencing of the hxA5 mutation, transformation mapping showed that hxA5 is comprised in the HindIII-EcoRV fragment contained in plasmid pBAN873X. This segment was amplified as described above from DNA extracted from strain SL453. The primers used were 5`-GAACTTTACCATGCCTC and 5`-GAGTCGTGTTTGTAGCG, which hybridize with sequences external to the two relevant restriction sites; the amplified band was cut with HindIII and EcoRV and cloned in Bluescript KS. Three independent clones were sequenced with identical results.

cDNA Clones

The 3`-end was determined from cDNA clones obtained by screening with pBAN816C (see ``Results''), a library made in the ZAPII vector (Stratagene) kindly provided by W. E Timberlake (Aramayo and Timberlake, 1990). The 5`-end was determined using the 5`-RACE (^5)kit (Life Technologies, Inc.) according to the recommendations of the manufacturer. The reverse transcription reaction was carried out with the oligonucleotide 5`-CGAAACGACAACCGTGCAAGC, which spans the first intron and thus only hybridizes with mature mRNA, and the amplification reaction with 5`-CCCATTCTTCAGTGAGTTGC and the ``anchor primer'' of the 5`-RACE kit. Amplification of the cDNA was carried out through 30 cycles of denaturation (1 min at 94 °C), annealing (1 min at 58 °C), and elongation (1 min at 74 °C) using Taq polymerase (Boehringer Mannheim) and a Bio-med GmbH thermocycler 60. For determination of the first intron, the reverse transcription reaction was carried out with the oligonucleotide 5`-GTCGATATCCAGTACAGC, and the amplification reaction (as described above) with 5`-GATGGAGGCATGGTAAAGC and the anchor primer of the 5`-RACE kit. For the second intron, the oligonucleotides 5`-GAGTCGTGTTTGTAGCG (for the reverse transcription reaction) and 5`-CTCGTGTTTCCAGAGCG and 5`-GAAGCTTTACCATGCCTC (for the amplification reaction) were used. For the determination of the third intron, we used the oligonucleotide 5`-GCTTGGTTGAGAAAGAGG for the reverse transcription reaction and 5`-GCTTGACCCTTGACACG and 5`-GAGAAGCTCAGTACACC for the amplification reaction. The amplified cDNA was cloned using the SureClone ligation kit (Pharmacia, Uppsala, Sweden) following the recommendations of the manufacturer.

Northern Blotting

About 15 µg of RNA was separated on a 1% agarose gel under the conditions described previously (Oestreicher and Scazzocchio, 1993). The hxA probe corresponded to the BglII-BglII fragment of 6.1 kbp (5) (see ``Results'').

Computer Methods

The handling, analysis, and translation of the sequences were accomplished with the DNA Strider 1.1 (Marck, 1988); comparisons were accomplished with the Pileup (of the University of Wisconsin Genetics Computer Group package) and BLAST (Altschul et al., 1990) programs using the facilities of the BLAST network service at the National Center for Biotechnology Information server asking for nonredundant data base (SwissProt, PIR, and the translation of most of the recent EMBL/GenBank nucleotide sequences) and using the BLOSUM62 matrix.

RESULTS AND DISCUSSION

Cloning of the hxA Gene

Strain CS2008 (see ``Experimental Procedures'' for full genotype), carrying both argB2 (resulting in a requirement for arginine) and hxA1 (a loss-of-function allele of the hxA gene) mutations, was transformed with the library constructed in plasmid pFB39 (see ``Experimental Procedures''), and growing colonies were selected on hypoxanthine as nitrogen source in the absence of arginine. One of the transformants did not require arginine, grew on hypoxanthine as nitrogen source, and carried all the other genetic markers of the parent strain. Southern blots showed that this transformant carried one plasmid integrated elsewhere than in the argB gene (data not shown). Uncut DNA extracted from the transformed strain was used to transform E. coli strain DH1. Three ampicillin-resistant clones were obtained, one of which carried a 28-kbp plasmid (pTA13C) that transformed the A. nidulans strain CS2008 for both the hxA1 and argB2 markers simultaneously at frequencies of >100 transformants/µg of DNA. The plasmids obtained from the other two clones were smaller and transformed strain CS2008 at a much lower frequency. It was thus likely that plasmid pTA13C carried an intact hxA gene, while the other two clones contained only a portion of the gene. Northern blots using plasmid pTA13C revealed, among other constitutive messages, a 4.5-kilobase mRNA inducible by 2-thiouric acid, as expected for the hxA mRNA. A 7-kbp SalI-EcoRI fragment internal to the pTA13C plasmid hybridized only with the putative hxA mRNA. As plasmids extracted from A. nidulans have gone through at least two recombination events, they can, and in some cases have been shown to, have rearrangements. Thus, a EMBL4 library (see ``Experimental Procedures'') was screened with plasmid pTA13C. A phage, LAN803, was obtained that has a restriction map overlapping with plasmid pTA13C. A 6.1-kbp BglII-BglII fragment, internal to the 7-kbp SalI-EcoRI fragment from phage LAN803, was subcloned into Bluescript KS to yield plasmid pBAN884. The fragment cloned in pBAN884 recognizes the same inducible mRNA as the original plasmid rescued from A. nidulans and was shown to transform strains carrying four different alleles of the hxA gene, hxA14 and hxA1 mapping at one end of the fine structure map (later shown to be the amino terminus), hxA102 mapping in the middle of the gene and corresponding to a substrate-binding site mutation (Scazzocchio and Sealy-Lewis, 1978), and hxA15 mapping at the opposite end of the fine structure map. Southern blots of a number of transformants showed the three types of integration events expected for a plasmid carrying the intact hxA gene: gene conversion, homologous single crossover, and heterologous integration events (data not shown). Fig. 1shows the restriction maps of plasmid pBAN884 and of a number of subclones obtained from the latter. The sense of transcription was determined by hybridization with single-stranded plasmids also indicated in Fig. 1.

Regulation of hxA Expression

Fig. 2shows that the hxA mRNA is inducible by the gratuitous inducer 2-thiouric acid, an analogue of the physiological inducer uric acid (Scazzocchio and Darlington, 1968), and that loss-of-function mutations in the uaY gene result in a low level of mRNA under both noninduced and induced conditions. uaY6 is a chain termination mutation in the amino terminus of the uaY gene. uaY205 is a 16-base pair deletion in the carboxyl terminus of the coding region of the gene (see ``Experimental Procedures''). The slight amount of induction seen in uaY205 may be due to residual transcriptional activation activity, as similar results were found for the xanthine dehydrogenase cross-reacting material in another carboxyl-terminal nonsense mutation (uaY207) (Scazzocchio, 1994). Fig. 2b shows that hxA transcription is repressible by 10 mM ammonium (urea, lane2), as expected from previous work in which the enzyme activity was measured (Scazzocchio and Darlington, 1968). To test the dependence of hxA transcription on the AreA factor, an areA600 mutation was used. This areA null mutant can grow only on ammonium or glutamine, both of which are repressing nitrogen sources. In Fig. 2(b and c; ammonium (amm.), lanes 1, 2, and 3), we have used 2.5 mM ammonium as sole nitrogen source. This is one-fourth of the usual concentration and for most other activities is only partly repressing. For hxA transcription, this concentration of ammonium affords the same repression as 10 mM. Thus, while it is very probable that full-rate transcription of hxA demands the AreA transcription factor, this cannot be formally shown. This is at variance with the uaZ and uapA genes, where a null mutation in the areA gene decreases transcription below the level found in the presence of 2.5 mM ammonium (Oestreicher and Scazzocchio, 1993; Suárez et al., 1991; Gorfinkiel et al., 1993).

Figure 2: Transcriptional regulation of the hxA gene. Northern blotting was performed as described under ``Experimental Procedures.'' The hxA probe was the 6.1-kbp BglII-BglII fragment (see Fig. 1) labeled with [alpha-P]dCTP using random hexanucleotide primers (Amersham Corp.). The same blots were also hybridized with the actin gene (Fidel et al., 1988) similarly labeled as a control of RNA loading. In all panels, urea indicates growth for 20 h on 5 mM urea as nitrogen source, and amm. indicates growth for 20 h on limiting ammonium (2.5 mM) as nitrogen source. Lanes 1, no additions; lanes 2, addition of both inducer and corepressor (27 µM 2-thiouric acid and 10 mM ammonium ion, respectively); lanes 3, addition of inducer. In a, we compare a uaY strain and a uaY205 strain. In b, two comparisons are made: first, a uaY6 strain with a wild-type (indicated as uaY) strain grown on urea as nitrogen source in lanes 1-3; second, we show the repressing effect of growth on limiting ammonium (2.5 mM) on the wild-type strain. In c, we compare a wild-type (indicated as areA) and an areA600 strain grown on limiting ammonium (2.5 mM) as sole nitrogen source. The wild type grown on urea under inducing conditions is also included as a standard (lane 3). Each panel corresponds to a different and separate experiment. In c (as in the other panels), all lanes come from one and the same Northern blot, but other lanes irrelevant to this experiment were cut out. To permit all relevant comparisons, the experiments shown in the three panels overlap partially. b and c are overexposed (in relation to a) to reveal more clearly the low amounts of xanthine dehydrogenase mRNA present under noninduced or repressed conditions. Thus, lanes 3 for the wild type (indicated as uaY and areA, respectively) are saturated, and this results in an apparently lower induced/noninduced ratio than in a.


Genomic and cDNA Sequence of the hxA Gene

We have sequenced the 5.5-kbp BglII-SacII genomic fragment included in plasmid pBAN884. Eventually, this fragment was shown to include the whole hxA gene (see below). The sequence strategy is shown in Fig. 1. cDNA clones were obtained by screening a ZAPII cDNA library with plasmid pBAN816C. Three independent polyadenylated clones were isolated (see below), none of them comprising the entire hxA cDNA, the longest being 1.7 kbp. These clones were sequenced. The obvious amino acid similarity between the A. nidulans hxA translated sequence and the cDNA sequences from a number of organisms suggested that three introns were present (see below). The positions and sizes of the introns were determined by polymerase chain reaction amplification of hxA cDNA using appropriate primers as detailed under ``Experimental Procedures'' and sequencing the clones so obtained. The transcription start site was determined by reverse transcription using the 5`-RACE protocol (data not shown; start site indicated in Fig. 3). The different cDNA clones overlap, and thus, the whole cDNA sequence was determined. The genomic sequence of the hxA gene is shown in Fig. 3.




Figure 3: hxA nucleotide sequence and deduced amino acid sequence. Noncoding regions and introns are presented in lower-case letters. The upper numbers to the right of the sequence indicate nucleotides, and the lower numbers indicate amino acids. The arrows are above the 5`-WGATAR sequences that are putative AreA-binding sites. The dashed line shows the TATA-like sequence. The pyrimidine-rich sequence is underlined. The verticals half-arrows indicate transcription initiation and termination points. Boldface type indicates the 5`- and 3`-splicing and lariat intron sites. The box corresponds to the AATAA sequence that is probably involved in RNA termination. The double-headed arrow indicates a sequence that has been implicated in mRNA termination (see ``Results and Discussion''). The mutational change in hxA5 is indicated above the nucleotide sequence. Also indicated are the HindIII and EcoRV sites that limit the fragment determined by transformation experiments to contain the hxA5 mutation and the ClaI sites that correspond to the limits of plasmid pBAN882C, which has been shown to contain the wild-type sequences corresponding to four substrate specificity mutations (see ``Results and Discussion'').


The size of the transcribed mRNA was estimated at 4.5 kilobases on Northern blots and is entirely contained in the 5.5-kbp fragment sequenced (data not shown; see below). A putative TATA box, 5`-TATTAA, is 37 nucleotides upstream from the start of transcription. Three 5`-WGATAR sequences that are potential AreA-binding sites (Fu and Marzluf, 1990; Merika and Orkin, 1993) are found upstream from the start of transcription. A pyrimidine-rich region precedes the start of transcription, as seen frequently in Ascomycetes. The different cDNA clones analyzed have poly(A) tails from 33 to 36 nucleotides long. The sequence 5`-AATAA upstream from the termination point of the mRNA could be the polyadenylation signal (Fitzgerald and Shenk, 1981; Hawkins, 1987). However, a 5`-CATGGTGAT sequence is also present; this sequence has also been implicated in mRNA termination (Upshall et al., 1986). The 5`-, 3`-, and lariat intron sequences show some agreement with the sequences previously proposed as consensus sequences (Ballance, 1986; Gurr et al., 1987).

The sequence surrounding the putative ATG codon shows an excellent agreement with other sequences described for lower eukaryotes, in particular for the uaZ and uapA genes. The codon usage does not differ significantly from the codon usage reported for most genes of A. nidulans (Lloyd and Sharp, 1991).

The translated peptidic sequence has 1363 amino acids. The RNA transcript of the hxA gene contains 4.425 nucleotides. The size of the mature mRNA, 4.284 nucleotides after subtraction of the intron sequences, compares well with the 4.2 kilobases estimated by preparative ultracentrifugation followed by identification by in vitro translation (Hanselman, 1984). The calculated M(r) of the polypeptide is 149,410. This compares well with the M(r) of the dimer estimated at 304,000 on nondenaturing gels (Lewis et al., 1978), but is larger than the value of 135,000 estimated for the monomer run on denaturing gels after immunoprecipitation (Hanselman, 1984). The pI of 5.86 estimated using the program MacVector (Kodak Scientific Imaging Systems, New Haven, CT) is similar to those of other xanthine dehydrogenases (Keith et al., 1987; Riley, 1989; Houde et al., 1989).

Formal Proof That the Gene Cloned and Sequenced Is hxA

Sequence comparisons (see below) leave no doubt that the isolated gene codes for a xanthine dehydrogenase. However, the far-fetched possibility that the gene we have cloned and sequenced is not hxA, but another gene coding for another, unknown xanthine dehydrogenase, remained open. The gene cloned is not hxnS (coding for purine hydroxylase II) (Scazzocchio, 1980), as Southern blots of DNA extracted from a strain carrying a large deletion covering hxnS and adjacent genes show no difference with DNA extracted from a wild-type strain when plasmid pBAN884 was used as a probe. Transformation to hxA of a number of hxA alleles by gene conversion (see above) confirms the identity of the cloned sequence with hxA. A number of transformation experiments showed that different restriction fragments, internal to the open reading frame, transform a number of hxA alleles to hxA. The physical positioning of each mutation located by this methodology is congruent with the order of mutations in the fine structure map. These results, together with the complete mutational analysis of the hxA gene, will be reported elsewhere, (^6)but one experiment providing formal proof that we have cloned and sequenced the hxA gene is described below. The hxA5 allele results in the total loss of enzyme activity and barely detectable amounts of cross reacting material (Darlington et al., 1965). (^7)It was located by transformation with subcloned hxA fragments in an interval between the HindIII site at position 1252 and the EcoRV site at position 1864 (Fig. 3). This was determined by transformation, as described under ``Experimental Procedures,'' with plasmid pBAN873X (which transforms this mutation to the wild-type phenotype, while none of the other plasmids shown in Fig. 1does) and a number of subclones derived from this plasmid (not shown in Fig. 1). The fragment was sequenced and showed to contain a C to T transition at position 1790, creating a UAG stop codon that results in a peptide only 369 amino acids long. This is indicated in Fig. 3.

Comparison of Eight Xanthine Dehydrogenase Peptidic Sequences: Domain Structure of the Xanthine Dehydrogenases

The cDNA sequences of a number of mammalian xanthine dehydrogenase genes (mouse, rat, and human) are available. Moreover, both the genomic and cDNA sequences of the genes form two species of Drosophila and of the fly Calliphora vicina are also available. The sequence of the translated HxA polypeptide is useful as an outclass for the mammalian and dipteran sequences. We can assume that sequences conserved in all three classes are important for the substrate specificity, the electron transport chain, or the dimerization of the enzyme. The comparison of the translated polypeptide sequences of all known xanthine dehydrogenases is shown in Fig. 4. Taking the D. melanogaster enzyme as a reference, the A. nidulans enzyme is the one that shows the lowest degree of similarity. This may be due to the fact that all the other enzymes are from metazoans and thus increases the functional significance of the conserved residues. There are, however, a number of changes, usually conservative, where the A. nidulans sequence is identical to either the mammalian (usually) or dipteran (less frequently) enzymes, when those are different among the latter groups. This might indicate which residue was present in the ancestral enzyme that preceded the divergence of fungi and metazoans.





Figure 4: Comparison of eight xanthine dehydrogenase peptidic sequences. The comparison has been carried out with the Pileup program of the University of Wisconsin Genetics Computer Group software and printed with the Prettybox program. Black, identical amino acids; dark and light gray, similar amino acids; white, nonconserved residues. Note that the program actually minimizes identities and similarities, as it compares sequences to a consensus sequence established when at least five out of the eight proteins have the same residue at one given position. For example, at position 26 of the A. nidulanssequence, we found an arginine in four sequences and glycine in four others. This is recorded as white (nonconserved) by the Prettybox program. In some places, slightly different alignments could be done by eye, which would increase similarities, but will create more gaps. An example of this can be found around positions 420-423 of the sequence of A. nidulans, where by creating a new gap, a universally conserved (except for H2) methionine will fall into place. The sequences are the following: A. nidulans (An), D. melanogaster (Dm) (Lee et al., 1987; Keith et al., 1987), D. pseudoobscura (Dp) (Riley, 1989), C. vicina (Cv) (Houde et al., 1989), rat liver (R) (Amaya et al., 1990), mouse liver (M) (Terao et al., 1992), human liver H1 (Ichida et al., 1993), and human liver H2 (Wright et al., 1993).


The amino acid identity of the A. nidulans xanthine dehydrogenase with the mammalian and insect enzymes is around 40-46% according to the organism or the alignment program used (see Fig. 4). The enzyme of A. nidulans is a few amino acids longer than all others. The additional amino acids are mainly at the amino terminus. It can be noticed that the amino terminus of the protein is highly variable, the similarity starting with a universally conserved phenylalanine at position 39 of A. nidulans and at position 8 of the D. melanogaster sequence.

The xanthine dehydrogenase of D. melanogaster has been shown to be peroxisomal (Beard and Holtzman, 1987). The putative peroxisomal localization signals of the enzyme (Gould et al., 1989), the AKL motif at positions 253-255 and AKI at positions 616-618, are conserved in the A. nidulans enzyme at positions 292-294 and 643-645, respectively.

The xanthine dehydrogenases have two [2Fe-2S] centers, described as an electron sink (Edmondson et al., 1973; Coughlan and Rajagopalan, 1980). The first center, revealed by analyzing the amino acid sequence with the PROSITE program (Bairoch, 1991), belongs to the same type found in the ferredoxin from a number of photosynthetic organisms, bacterial fumarate reductase, and eukaryotic succinic dehydrogenase. The general sequence of this motif is CX(4)CX(2)CX(n)C, where X is any amino acid and n equals 11 in succinic dehydrogenase, 29 in the ferredoxins, and 21 in all reported xanthine dehydrogenases. The second putative iron-sulfur center has been located between amino acids 78 and 176 in the sequence of the D. melanogaster xanthine dehydrogenase (Wootton et al., 1991). This corresponds to residues 108-206 in the A. nidulans sequence. An alignment between this region and a sequence of the iron-sulfur center of the ferredoxin of Clostridium pasteurianum has been proposed (Hughes et al., 1992b). All xanthine dehydrogenases have the sequence (H/N)G(S/T)QCGFCTP, while the sequence in the bacterial ferredoxin is NGKQQFCYS, showing a significant conservation around the second cysteine of the carboxyl-terminal iron-sulfur center.

The putative FAD-binding site cannot be precisely identified by sequence comparison. There are no obvious similarities to the consensus sequences described by Correll et al.(1993). The enzymatic data underlying the tentative identification of the FAD-binding site in the D. melanogaster enzyme (Hughes et al., 1992a) have now been withdrawn (Doyle and Bray, 1994). There is a cluster of missense mutations mapping in this region (residues 348, 353, and 357 in D. melanogaster) (Hughes et al., 1992a). This sequence is conserved in A. nidulans (residues 387-395). The A. nidulans enzyme shows three conservative changes in this region, one of which is shared with all the mammalian enzymes, with the exception of one of the human enzymes (H2 in Fig. 4). The fact that this conserved region maps just upstream from the NAD-binding site makes it likely that it is in fact the FAD-binding site. However, a direct demonstration has not yet been obtained.

The NAD-binding site has been chemically identified in the chicken xanthine dehydrogenase. The analogue 5`-p-fluorosulfonylbenzoyladenosine inactivates the enzyme by covalently binding to a tyrosine (Nishino and Nishino, 1987, 1989). This corresponds to tyrosine 429 in the sequence of A. nidulans. Excluding the aberrant human enzyme (H2 in Fig. 4), the following consensus sequence can be established starting at position 425 of the enzyme of A. nidulans: FFXGY*R(T/N)X(I/L)XPXH, where Y* denotes the tyrosine that is labeled by 5`-p-fluorosulfonylbenzoyladenosine in the chicken enzyme. All residues marked X differ between the mammalian and insect sequences. In every case, both the A. nidulans enzyme and the human H2 enzyme have a unique residue at these positions, which differs from both the mammalian and dipteran enzymes. This is of some relevance because Amaya et al.(1990) have argued that the environment of the Y* residue determines whether the conversion of a NAD-dependent form to an O(2)-dependent form is possible. No oxidase activity has been observed for the A. nidulans enzyme, either in its native form or following partial proteolysis (Mehra and Coughlan, 1989).

The domain involved in the fixation of the molybdenum cofactor has been tentatively identified by comparing the nitrate reductases from Arabidopsis thaliana and A. nidulans, the sulfite oxidases from rat and chicken liver, and the xanthine dehydrogenases from rat and D. melanogaster (Hughes et al., 1992b). The sequencing of the A. nidulans hxA gene permits the comparison of a nitrate reductase (Kinghorn and Campbell, 1989; Johnstone et al., 1990) and a xanthine dehydrogenase from the same organism. We have compared 11 available eukaryotic nitrate reductases, two sulfite oxidases, and the eight available xanthine dehydrogenases. We have derived a consensus sequence for each group of enzymes. This is shown in Fig. 5, with a suggested ``consensus'' sequence for the molybdenum cofactor-binding domain of eukaryotic molybdopterin-containing enzymes. The BLAST program revealed a clear similarity between the A. nidulans xanthine dehydrogenase and two prokaryotic enzymes, the aldehyde dehydrogenase from Acetobacter polyoxogenes (Tamaki et al., 1989) and the nicotine dehydrogenase from Arthrobacter nicotinovorans (GenBank accession number X75338). The putative molybdenum cofactor-binding domains of these two enzymes are also shown in Fig. 5. The nicotine dehydrogenase has been shown to contain molybdopterin (Freudenberg et al., 1988). There is no information about the cofactor complement of the aldehyde dehydrogenase (Tamaki et al., 1989), but the similarity data suggest that it may also contain a molybdenum cofactor. Brandsch (^8)has also observed similarities in the putative molybdenum cofactor-binding domain of the nicotine dehydrogenase and the eukaryotic xanthine dehydrogenases. Thoenes et al.(1994) have recently reported similarities between the aldehyde oxidoreductase from Desulfovibrio gigas and the metazoan xanthine dehydrogenases. They propose a large ``molybdopterin-binding domain'' that extends far beyond toward the carboxyl-terminal of what is proposed by us. A sequence similar to that shown in Fig. 5can also be found toward the amino terminus of their proposed molybdopterin-binding domain.

Figure 5: Putative molybdenum cofactor-binding domain. Ten sequences of nitrate reductase were aligned using the Pileup program of the University of Wisconsin Genetics Computer Group software including A. nidulans (Johnstone et al., 1990), Fusarium oxisporum (Diolez et al., 1993), Neurospora crassa (Okamoto et al., 1991), Ustilago maydis (Banks et al., 1993), Volvox carteri (Gruber et al., 1992), A. thaliana (Cheng et al., 1988), barley (Schnorr et al., 1991), Nicotiana tabacum (Vaucheret et al., 1989), rice (Cheng et al., 1989), and spinach (Prosser and Lazarus, 1990); the program Prettybox was utilized, and the consensus sequence was noted. The same procedure was applied for two sequences of sulfite oxidase: rat (Garrett and Rajagopalan, 1994) and chicken (Neame and Barber, 1989) liver. The alignment of the xanthine dehydrogenases is as described in the legend to Fig. 4. The BLAST program aligned the sequence of the xanthine dehydrogenase of A. nidulans with the aldehyde dehydrogenase of A. polyoxogenes (Tamaki et al., 1989) and the nicotine dehydrogenase of A. nicotinovorans. The sequence of the aldehyde oxidoreductase of D. gigas (Thoenes et al., 1994) was aligned by hand. The final alignment was made by hand. The double boxes represent the universally conserved amino acids; the single boxes represent the amino acids where we found conservative substitutions; and the numbers represent the residues between conserved amino acids. S.O. indicates the consensus sequence for sulfite oxidase (it corresponds to positions 137-214 of the rat enzyme). N.R. indicates the consensus sequence for the nitrate reductases (positions 72-157 of A. nidulans). XDH indicates the consensus sequence for the xanthine dehydrogenases (positions 773-862 of A. nidulans). Nic.D. indicates the sequence of the prokaryotic nicotine dehydrogenase (positions 203-288 of ndhC). Ald.D. indicates the prokaryotic aldehyde dehydrogenase (positions 368-452). Ald.O. indicates the prokaryotic aldehyde oxidoreductase (positions 369-446). Note that the later three enzymes do not have a cysteine that is conserved in all eukaryotic enzymes. cons. indicates the ``consensus of consensus'' for all eukaryotic sequences. corresponds to hydrophobic amino acids, and to asparagine or glutamine (we find an exception, serine in the U. maydis sequence); beta is equivalent to histidine or asparagine or glutamine, and alpha indicates an aspartic or glutamic acid. For 9 out of the 10 nitrate reductases, the sixth spacing between conserved residues (between beta and G) is of 16 or 19 amino acids; the exception is the nitrate reductase from N. crassa, which shows a spacing of 30 amino acids. Residues marked with asterisks are those where the H2 enzyme differs from other, conventional xanthine dehydrogenases.


Cofactor (cnx) and hxB mutations result in loss of xanthine dehydrogenase activity while maintaining NADH dehydrogenase activity (Scazzocchio, 1973; Lewis and Scazzocchio, 1977). But cofactor mutations also affect the stability of the A. nidulans xanthine dehydrogenase dimer, and in strains carrying such mutations, it is possible to detect on acrylamide gels the xanthine dehydrogenase monomer by its NADH dehydrogenase activity, while in hxB mutants, the stability of the dimer is not affected (Lewis and Scazzocchio, 1977). This makes a straightforward prediction on the phenotype that should be obtained by mutating the conserved residues in the putative molybdenum cofactor-binding domain.

A number of universally conserved stretches can be noticed carboxyl-terminal to the putative molybdenum cofactor-binding domain. Transformation experiments reported above have shown that at least some substrate specificity mutations map in this region. Thus, we propose that at least some of the conserved amino acids carboxyl-terminal to the putative molybdenum cofactor-binding domain participate in substrate binding. We observe that some residues that are conserved in rats, mice, human sequence H1, D. melanogaster, Drosophila pseudoobscura, and A. nidulans are not conserved in the other reported human sequences (H2 in Fig. 4). The sequence ERXXXH (A. nidulans positions 910-915) is universally conserved, except for H2 (Wright et al., 1993), which has a EMXXXK sequence. Mutagenesis experiments to be reported elsewhere have shown that conserved amino acids in the stretch ERXXXH at positions 910-915 of A. nidulans are actually involved in determining substrate specificity.^6 This sequence is within plasmid pBAN882C, which has been shown to contain the sequences altered in three substrate specificity mutations, hxA101, hxA102, and hxA143.

The proposed domain organization of the A. nidulans xanthine dehydrogenase is shown in Fig. 6. On the whole, the analysis of the A. nidulans sequence confirms the domain organization proposed previously (Hughes et al. 1992a, 1992b; Wootton et al., 1991), pinpoints a number of residues conserved in organisms as far apart as metazoans and fungi, and proposes a consensus for the molybdenum cofactor-binding domain.

Figure 6: Putative functional domains and intron position in the xanthine dehydrogenases. I and II indicate the iron-sulfur centers. F indicates amino acids involved in the binding of FAD, N indicates those involved in the binding of NAD, and M indicates amino acids involved in complexing the molybdenum cofactor according to the consensus sequence drawn in Fig. 5. S indicates a region of high similarity between all xanthine dehydrogenases corresponding to the region where the substrate specificity mutations map in the enzyme of A. nidulans (see ``Results and Discussion''). F and N are ``minimal'' domains (see ``Results and Discussion''), as the FAD and NAD domains may well overlap. The S region may be extended or limited as more substrate specificity mutations are located and sequenced. Short arrows, positions of introns in A. nidulans; double-headed arrows, positions of introns present in the three sequenced genes from Diptera; long single-headed arrow, intron present in both Drosophila species; long single-headed dashed arrow, intron present in D. pseudoobscura and C. vicina, but not in D. melanogaster.


The domain organization favored by us and others (Wootton et al., 1979; Hughes et al., 1992a, 1992b) on the basis of sequence comparisons may seem to conflict with the results obtained with the chicken xanthine dehydrogenase by Coughlan et al. (1979). These authors have shown that one product of subtilisin digestion carries the molybdenum-binding domain and the iron-sulfur centers, but not the FAD domain. The contradiction is only apparent, as this ``M(r) 65,000 domain'' obtained by subtilisin digestion has been shown to be composed of several peptides that stick together and are copurified. Such a complex could reflect the tertiary rather than the primary structure of the protein. Magnetic coupling studies of the native enzyme imply that the molybdenum and iron-sulfur domains lie near each other (8-14 Å) (Lowe et al., 1972; Lowe and Bray, 1978; Cottman and Buettner, 1979). This proposal is consistent with the more recent results of Amaya et al.(1990) on the rat liver enzyme.

The three introns interrupting the A. nidulans hxA gene are not in the same positions as the introns of the genes sequenced in Diptera. It would be particularly interesting to compare the A. nidulans intron positions with the mammalian ones, as on the whole, the A. nidulans enzyme is more similar to the mammalian than to the insect sequences. Unfortunately, no mammalian xanthine dehydrogenase genomic sequences are known. The introns of the A. nidulans hxA gene interrupt putative functional domains: the first and second interrupt the iron-sulfur domains, and the third interrupts the putative molybdenum cofactor-binding domain. This intron is in the beta-G interval (Fig. 5), which is the more variable interval in the proposed molybdenum cofactor-binding domain. The positions of the introns in the A. nidulans and insect enzymes are shown in Fig. 6.

While the human H2 enzyme shows marginally higher overall similarity to the D. melanogaster enzyme than to the A. nidulans enzyme, H2 differs from all other enzymes in several crucial sequences besides those in the putative NAD- and reducing substrate-binding sites (see above). The AKL putative peroxisomal domain is absent in this enzyme (residues 292-294 in the A. nidulans sequence). A hydrophobic residue (alanine in the mammalian and A. nidulans sequences and glycine in the dipteran sequences) in the putative FAD-binding domain is substituted with an arginine (this is the residue that is substituted with an aspartic acid in a mutant of D. melanogaster; see below). Three other universally conserved residues in this domain, a glutamine (position 388), a serine (position 393), and an isoleucine (position 395), are substituted with histidine, histidine, and aspartate, respectively. H2 is the only enzyme described that does not have a tyrosine in the putative NAD-binding domain (position 429 in the enzyme of A. nidulans); this residue is substituted with a cysteine. Other universally conserved residues, flanking this tyrosine, are also different in this human enzyme. On the other hand, the putative iron-sulfur centers and molybdenum cofactor-binding domains are well conserved in H2 (but see Fig. 5, which shows differences between H2 and all other xanthine dehydrogenases in the putative molybdenum cofactor-binding domain). We would like to propose that all the sequences, including the human enzyme H1 described by Ichida et al.(1993), but not the human enzyme H2 described by Wright et al.(1993), correspond to the same enzyme with identical or very similar substrate specificity. A number of enzymes called purine hydroxylases or, more imprecisely, ``aldehyde oxidases'' have been described. Krenitsky et al.(1972, 1974) have shown that the aldehyde oxidase from rat liver is very similar to the xanthine dehydrogenases, but differs in substrate specificity. These authors defined operationally aldehyde oxidases as enzymes able to hydroxylate 6-methylpurine, but not xanthine, while the xanthine oxidases (and by implication, the dehydrogenases) accept xanthine, but not 6-methylpurine as a substrate. By this criterion, mammals, including humans, have both a xanthine dehydrogenase (or oxidase) and an aldehyde oxidase. Moreover, most aldehyde oxidases, including the human liver enzyme, do not accept NAD as the oxidizing substrate (Krenitsky et al., 1974). This could well account for some of the sequence differences noted above. We propose that the human enzyme that has been cloned and sequenced by Wright et al. is another molybdenum-containing hydroxylase, related to but different from the classical xanthine dehydrogenases, and we anticipate that this enzyme will show different reducing and perhaps also oxidizing substrate specificities. As this enzyme has been isolated from a human liver cDNA, it may well be the human liver aldehyde oxidase described by Krenitsky et al.(1974).

It is interesting to look again at the missense D. melanogaster mutations now that the sequence of a non-metazoan enzyme is known. If we exclude the aberrant human H2 enzyme, all the missense mutations known (Hughes et al., 1992b) affect universally conserved residues. The only apparent exception is a conservative change, an alanine in the putative FAD-binding domain of the D. melanogaster enzyme (position 353) that is changed to aspartic acid in one of the mutants. At variance with the three diptera, the mammalian and A. nidulans enzymes have a glycine in this position.

The cloning and sequencing of the hxA gene of A. nidulans will allow the use of all the panoply of genetics and reverse genetics techniques to dissect the electron transport chain, the determinants of substrate specificity, the dimerization domains, and the region of interaction with post-translational modification functions, such as that coded by hxB. The strict conservation of the enzyme structure throughout evolution ensures that these studies will have general significance.

FOOTNOTES

*
This work was supported by CNRS, by the Université Paris-Sud, and by Grant SC1* CT92-0815 from the European Union. This paper is part of a series describing the molecular characterization of genes coding for enzymes and permeases of the purine degradation pathway of the fungus Aspergillus nidulans. The first two papers in this series are Gorfinkiel et al.(1993) and Oestreicher and Scazzocchio(1993). 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) X82827[GenBank].

§
To whom correspondence and reprint requests should be addressed. Tel.: 33-1-6941-6356; Fax: 33-1-6941-7808.

(^1)
H. M. Sealy-Lewis, S. Lee, G. Ong, and C. Scazzocchio, unpublished results.

(^2)
J. Kelly, S. Lee, and C. Scazzocchio, unpublished results.

(^3)
T. Suárez, M. Vieira Queiroz, N. Oestreicher, and C. Scazzocchio, manuscript in preparation.

(^4)
T. Suárez, N. Oestreicher, and C. Scazzocchio, unpublished results.

(^5)
The abbreviations used are: RACE, rapid amplification of cDNA ends; kbp, kilobase pair(s).

(^6)
A. Glatigny and C. Scazzocchio, manuscript in preparation.

(^7)
H. M. Sealy-Lewis and C. Scazzocchio, unpublished results.

(^8)
R. Brandsch, personal communication.

ACKNOWLEDGEMENTS

We thank R. C. Bray and R. Brandsch for providing data prior to publication and B. Labedan for invaluable help with the computer searches.

Addendum-While this article was being reviewed, a third cDNA human xanthine dehydrogenase sequence was published (Xu et al., 1994). The peptidic sequence is extremely similar (but not identical) to the enzyme sequence reported by Ichida et al. (1993) (H1 in Fig. 4). Xu et al. propose, as we do above, that the cDNA sequence determined by Wright et al.(1993) (H2 in Fig. 4) cannot correspond to a xanthine dehydrogenase. However, at variance with Xu et al., we propose (Fig. 5) that the cDNA sequence of Wright et al. represents an enzyme containing a molybdenum cofactor-binding domain.

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