Molecular properties of the lys1+ gene and the regulation of alpha-aminoadipate reductase in Schizosaccharomyces pombe.
The alpha-aminoadipate pathway for the biosynthesis of lysine is unique to fungi. Molecular properties of the cloned lys1+ gene and the regulation of the encoded alpha-aminoadipate reductase (AAR) were investigated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindIII-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 nested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-131 mutant phenotype was sequenced from the plasmid pLYS1D, and shown to 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 start codon. This ORF shared with the corresponding region of the isofunctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% similarity) overall, within which were smaller regions of marked sequence conservation. One such region coincided (95% identity) with a putative AMP-binding domain motif identified in the AAR of S. cerevisiae. In wild-type S. pombe, AAR activity from cells grown in lysine-supplemented 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 inhibition by lysine in vitro than the AAR of S. cerevisiae. These results show the effects of extensive evolutionary divergence on the structure and expression of a pivotal enzyme in the alpha-aminoadipate pathway. Presumably, delineated regions of strong sequence conservation correspond to discrete domains essential to AAR function.
Ford
R A
RA
Department of Microbiology, Miami University, Oxford, OH 45056, USA.
Bhattacharjee
J K
JK
D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D008239:Lysine / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D000445:Aldehyde Oxidoreductases; D050647:L-Aminoadipate-Semialdehyde Dehydrogenase; D008239:Lysine