CHARACTERIZATION OF A FUNGAL MALEYLACETOACETATE ISOMERASE GENE AND IDENTIFICATION OF ITS HUMAN HOMOLOG

We have previously used Aspergillus nidulans as a fungal model for hum an phenylalanine catabolism. This model was crucial for our characteri zation of the human gene involved in alcaptonuria, We use here an iden tical approach to characterize at the cDNA level the human gene for ma leylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unident ified structural gene of the phenylalanine catabolic pathway. We repor t here the first characterization of a gene encoding a MAAI enzyme fro m any organism, the A. nidulans maiA gene. maiA disruption prevents gr owth on phenylalanine (Phe) and phenylacetate and results in the absen ce 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 coil. maiA is clustere d 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. nidul ans and human enzymes of this pathway, we used the MaiA sequence in da ta base searches to identify human expressed sequence tags encoding it s putative homologues, Four such cDNAs were sequenced and shown to be encoded by the same gene, They encode a protein with 45% sequence iden tity to MaiA, which showed MAAI activity when expressed in E. coli, Hu man MAAI deficiency would presumably cause tyrosinemia that would be c haracterized by the absence of succinylacetone, the diagnostic compoun d resulting from fumarylacetoacetate hydrolase deficiency in humans an d fungi. Culture supernatants of an A. nidulans strain disrupted for m aiA are succinylacetone-negative but specifically contain cia and/or t rans isomers of 2,4-dioxohept-2-enoic acid. We suggest that this compo und(s) might be diagnostic for human MAAI deficiency.