From the Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain
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ABSTRACT |
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We have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway.
We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene. maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways.
Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli.
Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulans strain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2,4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency.
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INTRODUCTION |
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The catabolism of phenylalanine and tyrosine in humans is both of intrinsic and clinical interest. The enzymatic steps of this pathway were definitively established in the '50s by the work of Knox and colleagues (see Fig. 1A; Ref. 1). However, two of its structural genes remained uncharacterized. We recently used a novel approach based on the development of a fungal model to characterize one of them (2-4). Here we report our successful application of this approach to the characterization of the other and address by reverse genetics the consequences of the corresponding enzyme deficiency in our model organism.
An enzyme deficiency in any of the steps of this pathway causes in humans a known metabolic disease. For example, a deficiency in phenylalanine hydroxylase causes phenylketonuria (reviewed by Scriver et al. (5)). Enzyme deficiencies in four other steps (those labeled as II, III, and VI in Fig. 1A) cause different hypertyrosinemias (reviewed by Mitchell et al. (6)), and absence of homogentisate dioxygenase (IV, see Fig. 1A) causes alkaptonuria (4, 7). Although the historical interest in the later is notable as it enabled Archibald Garrod to coin the term of "inborn error of metabolism" (8, 9), the gene had not been characterized until recently (3, 4, 10). Crucial for the isolation and characterization of this gene was our establishment of a fungal model for human phenylalanine catabolism based on the filamentous ascomycete Aspergillus nidulans (2). We cloned its homogentisate dioxygenase gene (the first gene encoding this enzyme identified for any organism) and used its derived amino acid sequence as a probe to identify in similarity searches of the human expressed sequence, tag data base (EST)1 cDNAs encoding its human homologue (3).
Type 1 hereditary tyrosinemia (HT1, hepatorenal tyrosinemia, McKusick 276700) is the most severe disease in human Phe catabolism, affecting liver, kidney, and peripheral nerves. HT1 patients surviving infancy develop chronic liver disease with a high incidence of hepatocellular carcinoma (6). HT1 results from fumarylacetoacetate hydrolase (FAAH) deficiency (11). It is generally accepted that fumarylacetoacetate and its spontaneous reaction product, succinylacetone (the diagnostic compound of the disease), are toxic due to their considerable reactivity with key cellular molecules (6, 11), and fumarylacetoacetate has been shown to be mutagenic in Chinese hamster cells (12). In agreement with this, growth of an A. nidulans strain disrupted for the FAAH-encoding gene is prevented by phenylalanine even in the presence of an alternative carbon source (2). succinylacetone is accumulated in culture supernatants of this strain, illustrating the equivalent consequences of a FAAH deficiency in humans and A. nidulans (2).
The clinical consequences of a MAAI (MAAI, EC 5.2.1.2; step V in Fig. 1A) deficiency in humans are largely unknown. It is predicted that this deficiency should also lead to HT1, as maleylacetoacetate has similar reactivity to fumarylacetoacetate (for example, see Ref. 13). By contrast, it is thought that it should not result in the presence of succinylacetone in plasma and urine, as the latter compound is likely to be formed from succinylacetoacetate resulting from in vivo reduction of maleyl and fumarylacetoacetate (6, 11). Succinylacetoacetate is efficiently degraded by FAAH (1), and its hydrolysis would prevent succinylacetoacetone formation. Only one such succinylacetone-negative patient showing type 1 tyrosinemia with nondetectable levels of MAAI but normal levels of FAAH in liver has been described (14).
Mammalian MAAI has been little studied since its original characterization (1, 15, 16), possibly due, among other possible reasons, to the instability of the substrate (6). The gene encoding MAAI has not been cloned from any organism, and it is therefore the only structural gene of the Phe/Tyr degradation pathway that remains uncharacterized, precluding the analysis of the molecular basis of succinylacetone-negative type I tyrosinemia. Here we successfully use our fungal model to identify cDNAs encoding human MAAI. The liver enzyme requires glutathione (1, 15, 16) as does the equivalent bacterial enzyme that has been purified to homogeneity (17). Our characterization of fungal and human MAAI cDNAs revealed strong amino acid sequence identity of their derived protein sequences to glutathione S-transferases, in agreement with the proposed mechanism of the isomerization (18). We also extend the work of Edwards and Knox (16) and demonstrate MAAI activity by an in vitro complementation assay using extracts from a recombinant fungal strain deficient for MAAI. Notably, we detected no succinylacetone in culture supernatants of this strain.
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EXPERIMENTAL PROCEDURES |
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Fungal Strains, Media, and Growth Conditions--
A.
nidulans strains carried markers in standard use (19). A
biA1 strain was used as a source of cDNA and wild type
protein extracts. The biA1, methG1,
fahA strain has been described (2). A biA1,
methG1 strain was used as the wild type in growth tests. Standard media for A. nidulans (20) were used for strain
maintenance, growth tests, and transformation. Culture conditions
inducing high levels of expression of the fahA/maiA/hmgA
genes, which were routinely used to grow mycelia for protein
extraction, have been described (21).
Identification of the A. nidulans maiA Gene--
Genomic
maiA sequences were identified by Southern analysis of DNA
from EMBL4 clones carrying the fahA and hmgA
genes, using a subtracted cDNA probe representing genes induced by
phenylacetate ("plus" probe) and a cDNA probe from
glucose-grown mycelia ("minus" probe). A 2.5-kb EcoRI
fragment contiguous to the fahA transcription unit (see Fig.
1B) showed strong differential hybridization to the plus
probe. In addition, cDNA library screening using the F2 fragment
(see Fig. 1A) resulted in the isolation, in addition to
fahA cDNA clones, of a second class of cDNA clones
representing maiA transcripts. The cDNA library enriched
in transcripts induced by phenylacetate and the subtracted plus
cDNA probe have been described (2, 3). The A. nidulans
genomic library was a standard
EMBL4 library constructed from the
wild type strain. RNA isolation and Northern analysis followed (22).
Equal loading of the different samples was confirmed using an actin
probe.
Disruption of the maiA Gene--
Transformation followed Tilburn
et al. (23). For disruption of maiA we used a
4.2-kb linear DNA fragment in which the sequence between
maiA codons 140-226 had been replaced by a 3.2-kb
XbaI fragment carrying argB+. A
genomic fragment carrying maiA sequences from an
XbaI site at position 133 (relative to the initiation
codon) to an XhoI site at position +655 (relative to the
stop codon) was subcloned in pBS-SK+ (Stratagene).
Substitution of an internal 0.26-kb SalI-EcoRI fragment by the above 3.2-kb A. nidulans genomic fragment
(whose XbaI ends had been previously converted to
EcoRI and XhoI) removed maiA sequences
between codons 140 and 226 to yield pBS-
MAI. The transforming
fragment was isolated from this plasmid after digestion with
XbaI and XhoI.
Enzyme Assays--
Maleylacetoacetate, which is not commercially
available, was synthesized enzymatically from homogentisate (1, 15)
using homogentisate dioxygenase from A. nidulans extracts or
from Escherichia coli cells overexpressing the human enzyme.
The procedure used to obtain mycelial protein extracts from
fahA,
maiA, and wild type A. nidulans strains and the conditions for the homogentisate dioxygenase reaction have been described (21). For in vitro complementation assays, the initial homogentisate concentration was
100-125 µM. Maleylacetoacetate formation was monitored
spectrophotometrically at 330 nm. When the reaction reached a plateau
(with usually more than 80% of the substrate converted to
maleylacetoacetate), 150 µM reduced glutathione was added
to allow the MAAI-dependent isomerization of
maleylacetoacetate to fumarylacetoacetate (1, 16), which is then a
substrate for FAAH. Complementation of
maiA extracts was
used to detect MAAI activity in crude lysates of E. coli
cells overexpressing fungal or human MAAI, as described in the
corresponding figure legends.
Absorption Spectra of Intermediates-- Maleylacetoacetate was synthesized in separate reactions using 250-500 µM substrate and an excess of recombinant human HGO. When the reactions reached a plateau, they were stopped and deproteinized after the addition of 0.1 vol of 10% metaphosphoric acid, incubation for 10 min in ice, and centrifugation at 13,000 × g for 10 min. The supernatants were neutralized with KOH to pH 7.5-8 and immediately used in in vitro reactions in the presence of wild type or mutant A. nidulans protein extracts. These reaction mixtures were essentially as for homogentisate dioxygenase but contained GSH as a cofactor for MAAI. Reactions were carried out for 15 min at room temperature, deproteinized with metaphosphoric acid, and neutralized with KOH as above. The absorption spectra of these samples were determined and compared with those of duplicate reaction mixtures for which the final neutralization step was omitted.
Overexpression of Proteins in E. coli--
High levels of
protein expression were achieved using the pD1 vector (a gift of E. Espeso). This is a modified pET19b (Novagen) derivative that was
engineered to introduce a single BamHI site allowing
in-frame fusion of the desired coding region to an N-terminal His tag.
Details of this vector will be described elsewhere. Proteins overexpressed in this system carry the sequence
MGHHHHHHHHHHSSGHIDDDDKHMGS at their amino termini. The MaiA coding
region was amplified using the following pair of primers (underlined
sequences add or modify restriction sites):
5-CGGGATCCCCCGCACCGGTCAAGATCTC-3
(upper) and
5
-CGGAATTCAACACCTAAATTCCGTTGGTG-3
(lower). The fusion
protein contains the complete MaiA sequence with four further extra
residues (PAPL) between the above N-terminal tag and the MaiA
initiation methionine. The corresponding recombinant plasmid was
denoted pD1::MaiA. The human MAAI coding region was amplified
using EST 265310 (5
) as template and primers
5
-CAGGGATCCAAGCCCATCCTCTATTCC-3
(upper) and
5
-CAGGAATTCGGAGCTAGGCCCTC-3
(lower). The
recombinant gene fused the above N-terminal tag to residues 5-216 of
the protein. The corresponding plasmid was denoted
pD1::HSMAAI. A pD1::HSHGO plasmid (a gift from
M. C. Estébanez), driving high level expression of human
HGO, will be described elsewhere.
GC-MS Analysis of Culture Filtrates-- Fungal mycelia pregrown on 0.6% glucose (w/v) as the sole carbon source were transferred to appropriately supplemented minimal medium with 20 mM phenylacetate as the sole carbon source (see Ref. 21) and incubated for 20 h at 37 °C. Culture filtrates were ether-extracted and derivatized with bis(trimethylsilyl)trifluoroacetamide as described (2). TMS derivatives were analyzed by GC-MS in a fused silica capillary column SBP-1 (30 m × 0.25 mm; 0.2-mm film thickness) with a temperature program from 80 to 280 °C (4 °C/min), and a Q-MASS (Perkin-Elmer) mass detector. Identification of peaks was carried out by comparison of sample spectra with reference spectra from the NIST/EPA/NIH mass spectral data base.
DNA Sequencing--
Genes and cDNAs were sequenced using a
Dye Terminator Cycle sequencing kit (Perkin-Elmer) and Taq
FS DNA polymerase with universal and custom primers. Sequencing
reactions were resolved on an ABI Prism 377 automatic sequencer and
analyzed with the ABI analysis software (Version 3.1). Genomic and
cDNA versions of maiA and human EST cDNA clones
265310 (5), 290219 (5
), 683733 (5
), and 52677(5
) encoding human
MAAI were completely sequenced in both strands. cDNA clones of the
IMAGE consortium (25) were purchased from Genome Systems Inc., (St.
Louis, MO).2
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RESULTS |
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A Cluster of Three Genes Encoding Enzymes of the Common Part of the
A. nidulans PhAc/Phe Catabolic Pathway--
The ascomycete fungus
A. nidulans can use either Phe or PhAc as sole carbon source
(Fig. 1A). Both compounds were
catabolized to homogentisate, which was then converted to fumarate and
acetoacetate through the action of three enzyme activities, HGO, MAAI,
and FAAH. We have previously reported that the A. nidulans
fahA and hmgA genes encoding FAAH and HGO,
respectively, are closely linked and divergently transcribed from a
414-base pair intergenic region. fahA and hmgA
gene transcription is strongly inducible by PhAc (or its structural
relatives) or Phe and partially repressible by glucose. Neither of
these genes is expressed on glucose as the sole carbon source. No gene
encoding MAAI has yet been characterized from any organism. Southern
blot hybridization of EMBL4 phage clones carrying the
fahA and hmgA genes with a substracted cDNA probe representing transcripts induced by PhAc revealed the presence of
a third linked gene strongly hybridizing to this probe. As fahA and hmgA, this third gene, designated
maiA, was not expressed on glucose. Clustering of genes
encoding activities of the same catabolic pathway is not unusual in
A. nidulans. Genomic and cDNA nucleotide sequencing of
the region encoding this new transcript confirmed the presence of a
third gene 3
from fahA, transcribed in a tail-to-tail
orientation (Fig. 1B). The transcribed region contains an
intron-less ORF encoding a putative 230-residue polypeptide (Fig.
2) whose stop codon is 486 base pairs
downstream from that of fahA. The nucleotide sequence of
this three-gene cluster has been submitted to the
DDBJ/EMBL/GenBankTM data bases under accession number
AJ001836.
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A. nidulans maiA Encodes Maleylacetoacetate Isomerase-- Data base searches revealed that the predicted product of maiA shows strong amino acid sequence identity to glutathione S-transferases. For example, FASTA searches of the Swiss-Prot data base revealed that among the 30 protein sequences showing the highest alignment scores, 27 were glutathione S-transferases (data not shown). Similar results were obtained after searching the conceptual translation of EMBL + GenBankTM nucleotide sequence data bases with TBLASTN. The highest FASTA score corresponded to glutathione S-transferase 1 from Diantus caryophyllus, which showed 33.3% identity to MaiA in a 228-amino acid overlap including the complete sequence of both proteins (data not shown). The deduced molecular mass for MaiA (25,129 Da) is similar to the 25-kDa size of glutathione S-transferases.
Northern analysis showed that, as determined for fahA and hmgA, transcription of maiA was induced by either Phe or PhAc and was absent on glucose or gluconeogenic carbon sources (Fig. 3), strongly suggesting that maiA was a gene for Phe/PhAc catabolism and that its clustering with fahA and hmgA reflected its involvement in the same catabolic pathway. The only as yet unidentified gene encoding an enzyme essential for both Phe and PhAc catabolism is that encoding MAAI. The likely mechanism of this enzyme involves transfer of enzyme-bound GSH to C2 of maleylacetoacetate (26). Therefore, all the above data indicated that maiA might encode A. nidulans MAAI.
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Expression of A. nidulans MaiA in E. coli--
To definitively
establish that MaiA is A. nidulans MAAI, we expressed the
polypeptide as a fusion protein (see "Experimental Procedures") in
E. coli under the control of a T7 RNA
polymerase-dependent promoter. Promoter induction resulted
in the synthesis of a markedly abundant 32-kDa protein that was absent
from extracts of induced cells carrying the expression vector with no
insert. This protein, whose electrophoretic mobility was roughly
consistent with the Mr predicted for the MaiA
fusion protein, remained soluble upon clarification of the cell lysate
(data not shown). In vitro complementation of A. nidulans maiA and
fahA protein extracts described
above suggested the use of
maiA extracts in a coupled
enzyme assay for MAAI.
maiA extracts are unable to
transform homogentisate to fumarate and acetoacetate unless
supplemented with MAAI enzyme. Fig. 7
shows that an extract from E. coli cells overexpressing MaiA
complemented this MAAI deficiency in vitro. The coupled
action of bacterially expressed MAAI and endogenous A. nidulans FAAH resulted in the efficient degradation of
maleylacetoacetate. This coupled degradation obligately required GSH
and did not occur with an extract of E. coli cells carrying
the expression vector with no insert (Fig. 7).
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Analysis of A. nidulans Culture Supernatants for Diagnostic
Compounds of MAAI Deficiency--
An A. nidulans strain
disrupted for the gene encoding FAAH accumulates succinylacetone, the
diagnostic compound for HT1 (caused by FAAH deficiency) in the urine of
human patients. Growth of a fahA strain on lactose (which
is unaffected by the mutation) is strongly inhibited by Phe ((2); see
Fig. 4B). Phe toxicity in this mutant background is due to
fumarylacetoacetate and/or its spontaneous reaction product,
succinylacetone, which accumulate(s) as a result of the enzyme
deficiency. Notably, growth of a strain disrupted for maiA
was also inhibited by Phe (Fig. 4B), although clearly to a
lesser extent than that of the
fahA strain, strongly suggesting that different catabolites with distinct toxicities accumulate in each disrupted strain (but see "Discussion").
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Identification of Human EST Clones Encoding Homologues of A. nidulans MaiA-- We next used the fungal MaiA amino acid sequence to screen the EST data bases for human and murine ESTs encoding putative MaiA homologues, in analogy with the protocol already established for the AKU gene (3, 4). BLAST searches identified a number of these human ESTs. The 10 highest scores were obtained with the derived amino acid sequences of the following human ESTs (with the source of RNA for each EST cDNA clone in parentheses): 683733 (germinal B cells), 290219 (multiple sclerosis), 290775 and 265310 (melanocytes), 52677 (infant brain), 156401 (breast), 309975 (senescent fibroblasts), 240726 and 246479 (fetal liver/spleen), and 66e04 (skeletal muscle). These (partial) amino acid sequences showed more than 40% identity to that of A. nidulans MaiA, strongly suggesting that they represent its human homologue(s). The fact that only minor differences in the sequence (presumably resulting from automated sequencing errors) were found between these derived human proteins strongly suggests that all these cDNAs correspond to a single gene. Tissue-specific expression of such a gene does not appear to be as restricted as that of HGO (3, 4), and only two of the 10 cDNA clones represented liver transcripts (see above). In addition to the above human protein sequences, data base searches detected MaiA homology to derived protein products of mouse and Arabidopsis thaliana EST clones (not shown).
Molecular Characterization of Human cDNAs Encoding
Maleylacetoacetate Isomerase--
We fully sequenced four of the above
ESTs. (Fig. 9A). Nucleotide
sequencing showed that they were indeed encoded by the same gene,
despite the fact that they had been isolated from different tissues.
EST 265310 (5) (melanocytes) is the longest of these cDNAs. It is
1155 base pairs long (excluding the poly(A) tail) and contains a
216-codon ORF whose derived protein product (Mr 24,083) shows 45% identity in amino acid sequence to A. nidulans MAAI (Fig. 9B). This represents nearly
definitive evidence that this cDNA encodes a human MAAI (but see
below). The complete nucleotide sequence of this cDNA has been
submitted to DDBJ/EMBL/GenBankTM data bases (accession
number AJ001838) The 3
-UTR of this transcript was remarkably long (400 nucleotides, i.e. 30% of the transcript size). ESTs 290219 (5
) (multiple sclerosis lesions) and 683733 (5
) (germinal B-cells)
represented cDNAs incomplete at their 5
ends, starting at codons
18 and 49, respectively, of the human MAAI ORF. The precise site of
polyadenylation and the sequence of the 400-nucleotide 3
-UTR of EST
290219 were identical to those of the longest cDNA. Polyadenylation
of the 683733 cDNA occurred two nucleotides upstream of the above
site, but no other nucleotide sequence difference was observed either
in the 3
-UTR or in the coding region. Finally, we detected no
differences between the nucleotide sequences of EST 52677 (brain) and
EST 265310 up to position 1045, where the former is prematurely
polyadenylated as compared with the latter. This strongly suggests that
the 5
ends of these two transcripts represent a transcription start site for the human MAAI gene.
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DISCUSSION |
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We describe here the molecular characterization of
maiA, a gene encoding an enzyme of the common part of the
A. nidulans phenylalanine/phenylacetate pathways and provide
definitive biochemical and genetic evidence that this gene encodes a
maleylacetoacetate isomerase. This paper represents the first
characterization of a gene encoding MAAI from any organism. Compelling
evidence for the above conclusion can be summarized as follows: (i)
maiA is clustered with fahA and hmgA,
the two other structural genes of this common part of the pathways;
(ii) transcription of the gene is induced by phenylalanine or
phenylacetate as sole carbon sources; (iii) the deduced amino acid
sequence of its encoded protein shows identity to glutathione S-transferases, as expected for a MAAI enzyme; (iv)
disruption of the gene prevents growth on either Phe or phenylacetate;
(v) protein extracts from this disrupted strain convert homogentisate to maleylacetoacetate but cannot catabolize this compound further; (vi)
mixing maiA and
fahA extracts results in
reciprocal complementation of the corresponding enzyme deficiencies
required for maleylacetoacetate catabolism to fumarate and
acetoacetate; (vi) expression of maiA in E. coli
results in bacterial protein extracts showing MAAI activity. Two
technical developments were crucial to obtaining some of the above
evidence. First, we used either recombinant HGO enzyme (4) or fungal
extracts showing high HGO activity (21) to efficiently synthesize
maleylacetoacetate. Second, we used a complementation assay for MAAI
based on a protein extract from our A. nidulans strain
deleted for maiA. This extract converted homogentisate to
fumarate and acetoacetate only when supplied with GSH and a source of
MAAI.
We next used the MaiA-derived sequence to identify human, mouse, and plant ESTs encoding proteins showing high amino acid sequence identity to A. nidulans MAAI. Four such human cDNAs were fully sequenced and shown to encode a protein with 45% identity to MaiA. Although they were isolated from different tissues, these four cDNAs (and the other ESTs detected in our searches) almost certainly represent transcripts of the same gene. The protein encoded by this transcript(s) has MAAI activity when expressed in E. coli. Our electronic screening of the human EST data base would suggest that expression of this gene would be more ubiquitous than that of HGO, whose expression is largely restricted to liver, kidney, colon, small intestine, and prostate. This apparently less restricted pattern of expression might be related to its ability to use other compounds, in addition to maleylacetoacetate, as substrates (26), which might suggest a detoxification function (6).
Our characterization of human MAAI cDNAs represents the identification of the only as yet undescribed gene of the human Phe catabolic pathway. These results further confirm the validity of our fungal metabolic model and open the possibility of analyzing at the molecular level the predicted disease (a possible variant of HT1) resulting from MAAI deficiency. The incidence of this inborn error of metabolism is presently unknown, perhaps due to the absence of clear biochemical and/or molecular diagnostic criteria. A single patient with a putative MAAI deficiency has been reported in an abstract (14). Notably, this patient suffered from severe hepatorenal and brain damage. Our results with the fungal model show that the metabolite(s) accumulated as a result of a MAAI deficiency is indeed toxic for Aspergillus, but their toxicity is detectably lower than the toxicity of those accumulated as a result of a FAAH deficiency. Jorquera and Tanguay (12) have reported that, in contrast to fumarylacetoacetate, maleylacetoacetate was not mutagenic in Chinese hamster cells. We have not yet addressed if either of the above deficiencies is mutagenic in Aspergillus.
We detected no succinylacetone in culture supernatants of the A. nidulans maiA strain. This would be expected from the presumed origin of succinylacetone from decarboxylation of succinylacetoacetate, as normal levels of FAAH in this strain would degrade the latter (1).
Therefore, this absence of succinylacetone might, at least in part,
account for the lower Phe toxicity found in an A. nidulans strain deficient for MAAI as compared with a strain deficient for FAAH.
Analysis of culture filtrates of the A. nidulans
maiA strain specifically detected the presence of 4,6-dioxohept-2-enoic acid. This chemical structure would be consistent with maleylacetone (cis isomer) and/or fumarylacetone (trans
isomer). These isomers cannot be reliably distinguished by the
methodology used here. Taking into account the remarkable similarities
in the consequences of equivalent metabolic blocks in human and fungal
Phe catabolism, we suggest cis and/or trans
isomers of 4,6-dioxohept-2-enoic acid as possible diagnostic
compound(s) for MAAI deficiency in humans.
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ACKNOWLEDGEMENTS |
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We thank S. Rodríguez de Córdoba for critical reading of the manuscript, an anonymous reviewer for his/her careful checking of errors and useful suggestions, E. Reoyo for technical assistance, and A. Hurtado, V. Muñoz, and M. Fontenla for artwork.
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Note Added in Proof |
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An unpublished sequence recently submitted to the GenBankTM/EBI Data Bank with accession number U86529 and described as a cDNA encoding a human glutathione transferase Zeta 1 is the same as our cDNA sequence for human maleylacetoacetate isomerase.
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FOOTNOTES |
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* The work was supported by the Spanish Comisión Interministerial de Ciencia y Tecnología (BIO94/0932).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AJ001836, AJ001837, AJ001838.
To whom correspondence should be addressed: Tel.: 341-5611800 (ext. 4358); Fax: 341-5627518; E-mail: cibp173{at}fresno.csic.es.
1 The abbreviations used are: EST, expressed sequence tag; HT1, human type 1 hereditary tyrosinemia; GSH, reduced glutathione; PhAc, phenylacetate; FAAH, fumarylacetoacetate hydrolase; HGO, homogentisate dioxygenase; MAAI, maleylacetoacetate isomerase; TMS, trimethylsilyl; kb, kilobase(s); GC, gas chromatography; MS, mass spectrometry; ORF, open reading frame; UTR, untranslated region.
2 Details of EST libraries may be found in http://www-bio.llnl.gov/bbrp/image/humlib_info.html.
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REFERENCES |
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