MOLECULAR-PROPERTIES OF THE LYS1(-AMINOADIPATE REDUCTASE IN SCHIZOSACCHAROMYCES-POMBE() GENE AND THE REGULATION OF ALPHA)

The alpha-aminoadipate pathway for the biosynthesis of lysine is uniqu e to fungi. Molecular properties of the cloned lys1(+) gene and the re gulation of the encoded alpha-aminoadipate reductase (AAR) were invest igated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindII I-EcoRI fragment of S. pombe DNA, containing a functional lys1(+) gene and a promoter, was subcloned to make the 10.7-kb plasmid pLYS1H. A n ested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-1 31 mutant phenotype was sequenced from the plasmid pLYS1D, and shown t o contain an open reading frame (ORF) of 470 amino acids, preceded by putative POLII promoter elements (TATA and CCAAT box elements, and two potential yeast GCN4-binding motifs) within 368 bp upstream of the st art codon. This ORF shared with the corresponding region of the isofun ctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% s imilarity) overall, within which were smaller regions of marked sequen ce conservation. One such region coincided (95% identity) with a putat ive AMP-binding domain motifidentified in the AAR of S. cerevisiae. In wild-type S, pombe, AAR activity from cells grown in lysine-supplemen ted minimal or YEPD media was less than the activity of cells grown in minimal medium. The AAR of S. pombe was more sensitive to feedback in hibition by lysine in vitro than the AAR of S. cerevisiae. These resul ts show the effects of extensive evolutionary divergence on the struct ure and expression of a pivotal enzyme in the alpha-aminoadipate pathw ay. Presumably, delineated regions of strong sequence conservation cor respond to discrete domains essential to AAR function.