Alkaptonuria (AKU) occupies a unique place in the history of human genetics because it was the first disease to be interpreted as a mendelian recessive trait by Garrod in 1902. Alkaptonuria is a rare metabolic disorder resulting from loss of homogentisate 1,2 dioxygenase (HGO) activity. Affected individuals accumulate large quantities of homogentisic acid, an intermediary product of the catabolism of tyrosine and phenylalanine, which darkens the urine and deposits in connective tissues causing a debilitating arthritis. Here we report the cloning of the human HGO gene and establish that it is the AKU gene. We show that HGO maps to the same location described for AKU, illustrate that HGO harbours missense mutations that cosegregate with the disease, and provide biochemical evidence that at least one of these missense mutations is a loss-of-function mutation.

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 r ‫؍‬ 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 1 as the sole carbon source. The A. nidulans Phe catabolic pathway is notably similar to its human counterpart (Ferná ndez-Cañ ón and Peñ alva, 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.2 This PhAc pathway is absent in humans. 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 hom...

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.