(Received for publication, June 20, 1995; and in revised form, July 5, 1995)
From the
We report here the first characterization of a gene encoding a
homogentisate dioxygenase, the Aspergillus nidulans hmgA gene.
The HmgA protein catalyzes an essential step in phenylalanine
catabolism, and disruption of the gene results in accumulation of
homogentisate in broths containing phenylalanine. hmgA putatively encodes a 448-residue polypeptide (M = 50,168) containing 21 histidine and 23 tyrosine
residues. This polypeptide has been expressed in Escherichia coli as a fusion to glutathione S-transferase, and the
affinity-purified protein has homogentisate dioxygenase activity.
A. nidulans, an ascomycete amenable to classical and reverse genetic analysis, is a good metabolic model to study inborn errors in human Phe catabolism. One such disease, alkaptonuria, was the first human inborn error recognized (Garrod, A. E.(1902) Lancet 2, 1616-1620) and results from loss of homogentisate dioxygenase. Here we take advantage of the high degree of conservation between the amino acid sequences of the fungal and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human and plant homologues of HmgA. This is a significant advance in characterizing the genetic defect(s) of alkaptonuria and illustrates the usefulness of our fungal model.
The physiologically versatile filamentous ascomycete Aspergillus nidulans is able to grow on Phe or PhAc ()as the sole carbon source. The A. nidulans Phe
catabolic pathway is notably similar to its human counterpart
(Fernández-Cañón
and Peñalva, 1995). As in humans (Fig. 1),
Phe is degraded to homogentisate (2,5-dihydroxy-PhAc). The aromatic
ring is then cleaved by homogentisate dioxygenase to yield, after an
isomerization step, fumarylacetoacetate, which is split by
fumarylacetoacetate hydrolase into fumarate and acetoacetate (see Fig. 1). Aspergillus can also catabolize PhAc through
homogentisate after two sequential hydroxylation reactions in the
aromatic ring. (
)This PhAc pathway is absent in humans.
Figure 1: The Phe/PhAc pathway in A. nidulans. The enzymatic reactions involved in Phe catabolism are the same as in humans. Humans do not use the PhAc pathway.
Humans are very sensitive to defects in Phe catabolism. Loss-of-function mutations in structural genes of this pathway cause different metabolic diseases. Alkaptonuria is one such disease, resulting from loss of homogentisate dioxygenase (EC 1.13.11.15) (La Du et al., 1958). This moderately disabling disease, whose main clinical features are darkening of the urine, pigmentation of cartilages, and arthritis in adults, was the first inborn error of metabolism to be described (Garrod, 1902). However, the gene encoding homogentisate dioxygenase has not been characterized from humans or any other organism (see McKusick(1994)). Therefore, definitive evidence that the disease results from a loss-of-function mutation in this gene has not yet been obtained. Type I tyrosinaemia, resulting from fumarylacetoacetate hydrolase deficiency, is a different defect in human Phe catabolism with severe consequences. Our characterization of the fahA gene, encoding A. nidulans fumarylacetoacetate hydrolase, showed 47% identity at the amino acid level with its human homologue (Fernández-Cañón and Peñalva, 1995). Loss of this enzyme results in phenylalanine toxicity and extracellular accumulation of succinylacetone, a hallmark of the disease in the urine of human patients. The similarities in the overall organization of the Phe pathway, in the amino acid sequences for at least one enzyme, and in the consequences of equivalent genetic blocks between A. nidulans and humans prompted us to use this fungus as a model for certain metabolic aspects of human defects in Phe catabolism. Here we use this lower eukaryote to characterize, for the first time, a gene encoding a homogentisate dioxygenase and use its deduced amino acid sequence to identify its human and plant homologues.
Figure 2: Nucleotide sequence of the hmgA gene and amino acid sequence of its putative translation product. The nucleotide sequence shown was determined in both strands by sequencing a genomic and several overlapping cDNA clones covering the complete open reading frame. Coding and non-coding sequences are indicated in uppercase and lowercaseletters, respectively. Introns, all flanked by consensus donor and acceptor sequences, were determined by comparison of genomic and cDNA sequences. Putative lariat boxes, as determined by comparison to the consensus sequence described by Parker et al.(1987) in yeast, are underlined. Arrows indicate restriction enzyme cuts used to construct the disruption plasmid (see below).
Northern blot hybridization
experiments (Fig. 3) showed that this gene encodes an
1.7-kilobase message whose transcription pattern conforms to that
expected for a gene of Phe/PhAc catabolism. The gene was strongly
induced by PhAc, Phe, and certain monohydroxy derivatives of PhAc and
weakly induced by PhAc dihydroxy derivatives (on which the fungus grows
very poorly). Transcription was undetectable under carbon starvation
conditions or in the presence of either glucose or either of two
gluconeogenic carbon sources (acetate and glutamate). This pattern
strongly suggests that expression of this gene is specifically induced
by PhAc and Phe or by a common catabolite. Glucose only slightly
reduces induction by PhAc. This transcription pattern is identical to
that of the fahA gene
(Fernández-Cañón
and Peñalva, 1995). Indeed, Southern blot
hybridization experiments using DNA from purified phages from a
EMBL4 genomic library showed that this gene is closely linked to fahA, encoding A. nidulans fumarylacetoacetate
hydrolase. Nucleotide sequencing demonstrated that both genes were
transcribed divergently, their corresponding ORFs being separated by an
intergenic region of 415 base pair(s),
presumably
containing common elements controlling transcription of both genes.
Clustering of genes belonging to the same metabolic pathway is not
unusual in filamentous ascomycetes. This, together with the above
transcription data, strongly suggested that we had isolated a
previously uncharacterized gene of the Phe/PhAc catabolic pathway. The
deduced molecular mass of its encoded polypeptide is 50,168 Da, which
is very similar to that of 49 kDa estimated by SDS-polyacrylamide gel
electrophoresis for purified murine homogentisate dioxygenase (Schmidt et al., 1995). Thus, we suspected that this gene might encode
a fungal homogentisate dioxygenase. We describe below genetic and
biochemical evidence demonstrating that this is indeed the case, the Aspergillus homogentisate dioxygenase gene being designated hmgA.
Figure 3:
Transcription of hmgA is induced
by PhAc or Phe. Northern blot hybridization of total RNA isolated from
mycelia transferred to carbon sources indicated (- carbon
indicates no carbon source added; see ``Experimental
Procedures'' for concentrations of carbon sources). The probe was
a P-labeled cDNA containing the complete hmgA ORF. Loading in each lane was equal, as determined by
hybridization with an actin probe (not
shown).
Figure 4:
Disruption of the hmgA gene and
growth tests. Panel A, a plasmid containing a internal
fragment of hmgA was used in a transformation experiment to
disrupt the hmgA gene, as shown in the scheme (see
``Experimental Procedures'' for details). This integration
event results in two incomplete copies of hmgA, one lacking
the 101 C-terminal codons and the other lacking the promoter region,
the first exon, and part of the first intron. Two different
transformants having identical phenotypes had this integration event,
as established by Southern blot analysis using hmgA and argB probes (data not shown). Open boxes denote the argB gene; lined boxes denote the hmgA coding region (with three introns indicated); a wavy
line indicates A. nidulans coding sequences; and a straight line indicates vector DNA. Numbers above the hmgA coding region indicate codon position. Panel B,
growth phenotypes of the disrupted strain. Conidia of the hmgA
and of the hmgA::argB
strains were inoculated
on minimal medium plates with the indicated carbon sources (lactose at
0.05% (w/v), PhAc at 10 mM, and Phe at 25 mM). Plates
were incubated for 3 days at 37 °C before being
photographed.
Figure 5:
Disruption of hmgA results in
secretion of homogentisate and absence of homogentisate dioxygenase. Panel A, HPLC analysis of culture filtrates of the hmgA and the hmgA::argB
strains after transfer to defined medium with 25 mM Phe
as sole carbon source. The detector was set at the wavelengths
indicated. Homogentisate has a characteristic absorption maximum at 290
nm. Standards were as follows: I, 3,4-dihydroxyPhAc; II, homogentisate; III, 2,5-dihydroxybenzoate; IV, Phe; V, phenylpyruvate. Panel B,
homogentisate dioxygenase activity in mycelial extracts from the wild
type and the hmgA::argB
strains.
Figure 6:
Expression in E. coli and
purification of a GST::HmgA(9-448) fusion protein with
homogentisate dioxygenase activity. Panel A, assays of
homogentisate dioxygenase activity in crude and purified extracts from
bacteria carrying the plasmids indicated. The scheme shows relevant
features of the GST fusion genes. P indicates the promoter of pGEX2T. Panel B,
SDS-polyacrylamide electrophoresis of the GST::HmgA(9-448)
protein fusion expressed in E. coli. Samples were as follows: lane 1, size standards (Bio-Rad); lane 2, purified
fraction after elution with glutathione; lane 3, crude extract
after passage through the glutathione-Sepharose column; lane
4, crude bacterial extract; lanes 5 and 6, cells
lysed in sample buffer, 3 h after induction (lane 5) or
without IPTG induction (lane 6). Note the
76-kDa band
induced by IPTG, retained by the affinity column, and eluted with
glutathione (indicated by an arrow).
We noticed a
significant loss in the activity of the GST::HmgA (9-448) fusion
protein after purification by glutathione-Sepharose chromatography. The
specific activity of our purified fusion protein (Fig. 6A) is 7.5 times higher than that reported for
murine homogentisate dioxygenase (Schmidt et al., 1995). These
figures represent a minimal estimation for the specific activity of the Aspergillus enzyme, as our protein has been produced in a
heterologous host and we cannot exclude a negative effect of the
N-terminal GST moiety on enzyme activity. Our purified GST::HmgA
(9-448) fusion protein showed no activity when the reaction was
carried out in the absence of Fe ions, as described
for purified murine homogentisate dioxygenase (Schmidt et al.,
1995). In contrast, the enzyme was fully active in the absence of
ascorbate.
Figure 7: Identification of human and plant homologues of the hmgA gene. Sequence comparison between HmgA and deduced polypeptides encoded by human and plant ESTs. A, human 5`-ESTs. Identical residues are shown below the indicated regions of HmgA. Deduced sequences encoded by ESTs were grouped in two pairs, each corresponding to a different region of HmgA. It should be noted that sequences codified by T55939 and T27323 differ in three positions (residues in boldface (indicated by boxes); see also text). Conserved residues are not indicated for clarity. Residues marked as X represent ambiguities in the nucleotide sequence of the EST. Numbers on the right and leftends of HmgA sequences denote the positions of the amino acid residues in the protein. Numbers (in parentheses) on the leftside of each EST polypeptide refer to the nucleotide position of the first translated codon in its corresponding DNA sequence. In several cases, frameshifting allows alignments with significant sequence conservation, possibly reflecting sequencing errors in ESTs. Therefore, deduced sequences corresponding to different reading frames (indicated on the right) of the same 5`-EST are shown when necessary. Parentheses indicate regions of deduced sequence with no evident similarity in any of the three EST reading frames. Also indicated on the right are the GenBank accession numbers and the source of cDNA. The liver spleen library is a mixed library. B, plant 5`-ESTs. Sequence comparisons were shown as in A, indicating the organism from which the library was constructed.
The remaining three ESTs were from plants, two being from Arabidopsis. Their deduced polypeptides showed 44 and 43% identity to non-overlapping HmgA regions close to the N- and C-terminal regions, respectively. The polypeptide encoded by the third plant EST (from Ricinus communis) has 36% identity to the above near C-terminal HmgA region.
We describe here the first characterization in any organism of a gene encoding an homogentisate dioxygenase, the A. nidulans hmgA gene. Homogentisate dioxygenase activity is strongly induced in mycelia by Phe or PhAc, and hmgA cDNA clones were easily isolated using a subtracted cDNA probe from a library enriched in cDNAs for PhAc-induced transcripts. hmgA is essential for growth on Phe (or PhAc) as sole carbon source. This supports the A. nidulans Phe (PhAc) catabolic pathway as shown in Fig. 1. Disruption of the gene results in complete absence of homogentisate dioxygenase activity. Therefore, the disruption created here is almost certainly a null allele. Due to this defect, this strain, when supplied with Phe or PhAc, secretes homogentisate, which is readily oxidized to yield a reddish pigment, eventually turning dark brown. The complete absence of enzyme activity in the disruption strain together with the absence of cross-hybridizing bands in genomic Southern blots strongly indicates that A. nidulans contains a single gene encoding homogentisate dioxygenase.
Mammalian homogentisate dioxygenases contain weakly
bound ferrous ions that are required for activity (see Schmidt et
al.(1995) and references therein). A 70% reduction in activity was
also observed when crude Aspergillus extracts were
assayed in the absence of Fe
ions, and the activity
of the purified GST::HmgA (9-448) fusion protein showed an
absolute requirement for these ions. The deduced HmgA polypeptide
contains 21 His and 23 Tyr residues. Some of these residues might be
involved in binding iron, as demonstrated for protocatechuate
3,4-dioxygenase (Ohlendorf et al., 1988).
The deduced HmgA sequence was used to identify human ESTs potentially encoding a homologue(s) of the fungal gene. The high similarity detected at the amino acid level establishes that human genes corresponding to these ESTs are indeed hmgA homologues. The EST-encoded amino acid sequences are classified in two groups, each corresponding to a different region of HmgA. Therefore, our results do not establish the existence of a single human gene encoding homogentisate dioxygenase.
Three of these ESTs were isolated from liver or liver/spleen cDNA libraries. Alkaptonuria results from loss of homogentisate dioxygenase, as demonstrated in both liver and kidney extracts. Our preliminary identification of cDNAs for homogentisate dioxygenase, nearly a century after alkaptonuria was recognized by Garrod(1902) as an inborn error of metabolism, represents a significant advance in the characterization of the human gene and further illustrates the validity of our fungal metabolic model for disorders in human Phe metabolism (Fernández-Cañón and Peñalva, 1995). Definitive evidence that alkaptonuria results from loss-of-function mutation(s) in the homogentisate dioxygenase gene will require mapping of this gene to chromosome 3q2 (the location for alkaptonuria (Pollak et al., 1993; Janocha et al., 1994)) and identification of mutations in patients with the syndrome. Finally, availability of the human gene will allow examination of tissue-specific expression. One of the ESTs identified here was isolated from pancreatic islet cDNA. Homogentisate dioxygenase activity in this tissue has not been reported previously.
We have previously reported a class of mutations (suAfah)
suppressing the effects of a complete fumarylacetoacetate hydrolase
deficiency in A. nidulans (Fernández-Cañón
and Peñalva, 1995). The phenotype of these
suppressor mutations is indistinguishable of that caused by hmgA::argB mutation (i.e. loss of homogentisate dioxygenase and secretion of homogentisate),
and they do not complement the disruption in diploids. These suAfah mutations are therefore hmgA
alleles
(Fernández-Cañón
and Peñalva, 1995). This suppression led us to
propose that alkaptonuria would prevent the lethal effects of human
type I tyrosinaemia by blocking the pathway upstream of
fumarylacetoacetate hydrolase. Identification of mammalian homologues
of hmgA reported here will facilitate testing this hypothesis
in animal models.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].