Ford R A, Bhattacharjee J K
Department of Microbiology, Miami University, Oxford, OH 45056, USA.
Curr Genet. 1995 Jul;28(2):131-7. doi: 10.1007/BF00315779.
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.
赖氨酸生物合成的α-氨基己二酸途径是真菌所特有的。在裂殖酵母粟酒裂殖酵母中研究了克隆的lys1⁺基因的分子特性以及编码的α-氨基己二酸还原酶（AAR）的调控。粟酒裂殖酵母DNA的一个5.2 kb HindIII-EcoRI片段，包含一个功能性lys1⁺基因和一个启动子，被亚克隆以构建10.7 kb的质粒pLYS1H。从质粒pLYS1D中对一个与lys1-131突变体表型互补的1.778 kb嵌套HindIII-EcoRI DNA片段进行了测序，结果显示其包含一个470个氨基酸的开放阅读框（ORF），在起始密码子上游368 bp内有推定的POLII启动子元件（TATA和CCAAT框元件，以及两个潜在的酵母GCN4结合基序）。这个ORF与酿酒酵母同功能AAR的相应区域总体上有49%的氨基酸同一性（62%的相似性），其中有较小区域具有明显的序列保守性。其中一个这样的区域与在酿酒酵母AAR中鉴定出的推定AMP结合结构域基序一致（95%同一性）。在野生型粟酒裂殖酵母中，在补充赖氨酸的基本培养基或YEPD培养基中生长的细胞的AAR活性低于在基本培养基中生长的细胞的活性。粟酒裂殖酵母的AAR在体外比酿酒酵母的AAR对赖氨酸的反馈抑制更敏感。这些结果显示了广泛的进化差异对α-氨基己二酸途径中一种关键酶的结构和表达的影响。据推测，明确的强序列保守区域对应于AAR功能所必需的离散结构域。
