Urano Y, Manabe T, Noji M, Saito K
Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, 263-8522, Chiba, Japan.
Gene. 2000 Oct 31;257(2):269-77. doi: 10.1016/s0378-1119(00)00399-1.
The plants belonging to the genus Allium are known to accumulate sulfur-containing secondary compounds that are derived from cysteine. Here, we report on molecular cloning and functional characterization of two cDNAs that encode serine acetyltransferase and cysteine synthase from A. tuberosum (Chinese chive). The cDNA for serine acetyltransferase encodes an open reading frame of 289 amino acids, of which expression could complement the lacking of cysE gene for endogenous serine acetyltransferase in Escherichia coli. The cDNA for cysteine synthase encodes an open reading frame of 325 amino acids, of which expression in the E. coli lacking endogenous cysteine synthase genes could functionally rescue the growth without addition of cysteine. Both deduced proteins seem to be localized in cytosol, judging from their primary structures. Northern blot analysis indicated that both transcripts accumulated in almost equal levels in leaves and root of green and etiolated seedlings of A. tuberosum. The activity of recombinant serine acetyltransferase produced from the cDNA was inhibited by L-cysteine, which is the end-product of the pathway; however, the sensitivity to cysteine (48.7 microM of the concentration for 50% inhibition, IC(50)) was fairly low compared with that of previously reported serine acetyltransferases ( approximately 5 microM IC(50)) from various plants. In A. tuberosum, the cellular content of cysteine was several-fold higher than those in Arabidopsis thaliana and tobacco. This higher concentration of cysteine in A. tuberosum is likely due to the lower sensitivity of feedback inhibition of serine acetyltransferase to cysteine.
葱属植物已知会积累源自半胱氨酸的含硫次生化合物。在此，我们报告了从韭菜中克隆和鉴定编码丝氨酸乙酰转移酶和半胱氨酸合酶的两个cDNA的分子克隆及功能特性。丝氨酸乙酰转移酶的cDNA编码一个289个氨基酸的开放阅读框，其表达可以弥补大肠杆菌中内源性丝氨酸乙酰转移酶的cysE基因缺失。半胱氨酸合酶的cDNA编码一个325个氨基酸的开放阅读框，其在缺乏内源性半胱氨酸合酶基因的大肠杆菌中的表达能够在不添加半胱氨酸的情况下功能性地挽救其生长。从它们的一级结构判断，这两种推导蛋白似乎都定位于细胞质中。Northern印迹分析表明，在韭菜绿色和黄化幼苗的叶片和根中，这两种转录本的积累水平几乎相等。由该cDNA产生的重组丝氨酸乙酰转移酶的活性受到该途径的终产物L-半胱氨酸的抑制；然而，与先前报道的来自各种植物的丝氨酸乙酰转移酶（IC50约为5 microM）相比，其对半胱氨酸的敏感性（50%抑制浓度为48.7 microM，IC50）相当低。在韭菜中，半胱氨酸的细胞含量比拟南芥和烟草中的高出几倍。韭菜中较高浓度的半胱氨酸可能是由于丝氨酸乙酰转移酶对半胱氨酸的反馈抑制敏感性较低。
