THE ASPERGILLUS-NIDULANS LYSF GENE ENCODES HOMOACONITASE, AN ENZYME INVOLVED IN THE FUNGUS-SPECIFIC LYSINE BIOSYNTHESIS PATHWAY

In filamentous fungi, lysine is synthesized via the alpha-aminoadipate pathway. In order to gain insight into this fungus-specific pathway ( to date, no genes for enzymes of this pathway in filamentous fungi hav e been cloned) the lysine auxotrophic mutant LysF88 of Aspergillus nid ulans was studied. HPLC and H-1-NMR analyses revealed that LysF88 accu mulated homocitric acid in the culture supernatant. In addition, both the LysF88 mutant strain and LysF deletion strain (LysFKO) described h ere showed hardly any homoaconitase activity, indicating that lysF enc odes homoaconitase. The lysF gene was cloned by complementation of the LysF88 mutant and sequenced. It has a size of 2397 bp, including a si ngle intron of 72 bp. The two exons encode an open reading frame (ORF) of 2325 bp. The calculated M-r of the homoaconitase protein (775 amin o acids) is 83 943. A major and a minor transcript begin at positions -28 and -32, respectively. The 3' end of the lysF cDNA showed a poly(A ) tail commencing at position + 2647 following a 250 bp untranslated r egion after the lysF stop codon. A putative polyadenylation signal seq uence (TATAAA) is located 49 bp upstream of the polyadenylation site. Computer analysis revealed 55% amino acid sequence identity between th e products of the putative homoaconitase ORF of A. nidulans and that o f the recently sequenced homologous Saccharomyces cerevisiae. The simi larity was particularly obvious in a region of cysteine residues, whic h are characteristic of an iron-sulfur cluster, implying that homoacon itase contains such a cluster. The homoaconitases of A. nidulans and S . cerevisiae share only 20% sequence identity with S. cerevisiae aconi tase. The pH optimum for the activity of A. nidulans homoaconitase in 0.1 M potassium phosphate buffer is between pH 8.1 and pH 8.6. Homoaco nitase exhibited an apparent K-m of 1.1 mM toward homoisocitric acid. The specific activity of homoaconitase was reduced by up to six-fold i n mycelia grown in the presence of L-lysine, suggesting that it is reg ulated by lysine.