Phartiyal Pallavi, Kim Won-Seok, Cahoon Rebecca E, Jez Joseph M, Krishnan Hari B
Department of Agronomy, University of Missouri, Columbia, 65211, USA.
Arch Biochem Biophys. 2006 Jun 1;450(1):20-9. doi: 10.1016/j.abb.2006.03.033. Epub 2006 May 8.
Soybeans are a rich source of protein and a key feed ingredient in livestock production, but lack sufficient levels of cysteine and methionine to meet the nutritional demands of swine or poultry as feed components. Although engineering the sulfur assimilatory pathway could lead to increased sulfur-containing amino acid content, little is known about this pathway in legumes. Here, we describe the cloning and characterization of soybean ATP sulfurylase (ATPS), which acts as the metabolic entry point into the sulfur assimilation pathway. Analysis of the ATPS clone isolated from a soybean seedling cDNA library revealed an open-reading frame, encoding a 52 kDa polypeptide with an N-terminal chloroplast/plastid transit peptide, which was related to the enzymes from Arabidopsis, potato, human, and yeast. Soybean ATP sulfurylase was expressed in Escherichia coli and purified to apparent homogeneity. Based on gel-filtration chromatography, the enzyme functions as a 100 kDa homodimer. Analysis of genomic DNA by Southern blotting revealed that multiple genes encode ATP sulfurylase in soybean. Analysis of the transcript profiles retrieved from a soybean EST database indicated that ATP sulfurylase mRNA was most abundant in root tissue. Cold treatment induced mRNA accumulation and enhanced the specific activity of ATP sulfurylase in root tissue. Northern blot analysis indicated a decline in the ATP sulfurylase transcript levels during seed development. Likewise, ATP sulfurylase specific activity also declined in the later stages of seed development. Increasing the expression levels of this key enzyme during soybean seed development could lead to an increase in the availability of sulfur amino acids, thereby enhancing the nutritional value of the crop.
大豆是蛋白质的丰富来源，也是家畜生产中的关键饲料成分，但半胱氨酸和蛋氨酸水平不足以满足猪或家禽作为饲料成分的营养需求。尽管对硫同化途径进行工程改造可能会增加含硫氨基酸的含量，但对于豆科植物中的这条途径知之甚少。在此，我们描述了大豆ATP硫酸化酶（ATPS）的克隆和特性，它是硫同化途径的代谢入口点。对从大豆幼苗cDNA文库中分离出的ATPS克隆进行分析，发现了一个开放阅读框，编码一个52 kDa的多肽，其N端有叶绿体/质体转运肽，与拟南芥、马铃薯、人类和酵母中的酶相关。大豆ATP硫酸化酶在大肠杆菌中表达并纯化至表观均一。基于凝胶过滤色谱分析，该酶以100 kDa同型二聚体的形式发挥作用。通过Southern印迹分析基因组DNA表明，大豆中有多个基因编码ATP硫酸化酶。对从大豆EST数据库中检索到的转录谱进行分析表明，ATP硫酸化酶mRNA在根组织中最为丰富。冷处理诱导根组织中mRNA积累并增强ATP硫酸化酶的比活性。Northern印迹分析表明，种子发育过程中ATP硫酸化酶转录水平下降。同样，种子发育后期ATP硫酸化酶的比活性也下降。在大豆种子发育过程中提高这种关键酶的表达水平可能会增加硫氨基酸的可利用性，从而提高作物的营养价值。
