MOLECULAR CHARACTERIZATION OF A GENE ENCODING A HOMOGENTISATE DIOXYGENASE FROM ASPERGILLUS-NIDULANS AND IDENTIFICATION OF ITS HUMAN AND PLANT HOMOLOGS

We report here the first characterization of a gene encoding a homogen tisate dioxygenase, the Aspergillus nidulans hmgA gene. The HmgA prote in catalyzes an essential step in phenylalanine catabolism, and disrup tion of the gene results in accumulation of homogentisate in broths co ntaining phenylalanine. hmgA putatively encodes a 448-residue polypept ide (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 homo gentisate 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, alkapto nuria, was the first human inborn error recognized (Garrod, A. E. (190 2) Lancet 2, 1616-1620) and results from loss of homogentisate dioxyge nase. Here we take advantage of the high degree of conservation betwee n the amino acid sequences of the fungal. and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human an d plant homologues of HmgA. This is a significant advance in character izing the genetic defect(s) of alkaptonuria and illustrates the useful ness of our fungal model.