title: Characterization of a fungal maleylacetoacetate isomerase gene and identification of its human homologue.
abstract: 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.
authors.affiliation: Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Fernández-Cañón
authors.forename: M A
authors.initials: MA
authors.lastname: Peñalva
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology*; D001483:Base Sequence; D018076:DNA, Complementary; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D017386:Sequence Homology, Amino Acid; D019745:cis-trans-Isomerases / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D019745:cis-trans-Isomerases; C021531:maleylacetoacetate isomerase