Identification and characterization of a pyridoxal 5 '-phosphate phosphatase in tobacco plants
Identification and characterization of a pyridoxal 5 '-phosphate phosphatase in tobacco plants
复制标题
烟草植物中吡哆醛 5'-磷酸磷酸酶的鉴定和表征
DOI：
10.1016/j.plantsci.2018.10.014
复制
发表时间：
2019
期刊：
影响因子：
5.2
通讯作者：
Huang LongQuan
中科院分区：
文献类型：
--
作者：
Huang ShuoHao;Liu Jing;Zhou Jie;Zhang JianYun;Huang LongQuan
5‘-磷酸吡哆醛(PLP)是维生素B6的催化活性形式，是许多生化转化的重要辅因子。PLP也是一种活性很强的分子，维持低水平游离PLP最成熟的机制是它被磷酸酶去磷酸化。在我们之前的研究中，从烟叶中提取的粗酶在最适pH为5.5的条件下能够快速地降解PLP。以PLP为底物，从烟叶中分离纯化了一种新型酸性磷酸酶，并对其性质进行了研究。植物中是否存在PLP专一性的磷酸酶尚不清楚。在本研究中，鉴定出一个与人PLP磷酸酶序列有34.72%同源性的克隆。表格和特征。该基因编码319个氨基酸残基的多肽，重组酶纯化。在pH为7.5时，大肠杆菌对PLP的催化活性最高。该酶的最适pH、金属需要量、最适底物和抑制剂等性质与人PLP磷酸酶相似。亚细胞定位分析表明，PLP磷酸酶主要定位于叶绿体。我们利用植物RNA干扰技术下调了基因的表达，发现下调对维生素B6代谢酶编码基因的转录有更大的影响。我们的研究进一步表明，PLP磷酸酶在维持植物叶绿体内PLP动态平衡方面起着重要作用。
Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an important cofactor for many biochemical transformations. PLP is also a very reactive molecule, and the most well-established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. In our previous study, the crude enzyme extract from tobacco leaves rapidly hydrolyzed PLP at a pH optimum of 5.5. Using PLP as a substrate, a novel acid phosphatase was purified from tobacco leaves and characterized. Whether there is a PLP specific phosphatase in plants is still unknown. In this study, a cDNA clone sharing 34.72% homology with human PLP phosphatase sequences was identified fromN. tabacumand characterized. The cDNA encodes a polypeptide of 319 amino acid residues, and the recombinant enzyme purified fromE. coliexhibited maximum catalytic activity for PLP at pH 7.5. The properties of the purified enzyme, including pH optimum, metal requirement, optimum substrate and inhibitors were similar to those of human PLP phosphatase. Subcellular localization analysis showed that the PLP phosphatase is mainly located in chloroplast. We down-regulated the gene expression with plant RNA interference technology and found that the down-regulation has a greater impact on the transcription of genes encoding vitamin B6metabolic enzymes. Our study further suggested that the PLP phosphatase plays an important role for maintaining PLP homeostasis within the chloroplast in plants.