Noda Miki, Nakamura Mika, Takamiya Ryuichi, Tamura Takashi, Ito Toshiyuki, Kodama Hiroaki
Graduate School of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8510, Japan.
Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Biochim Open. 2016 Feb 8;2:24-32. doi: 10.1016/j.biopen.2016.01.002. eCollection 2016 Jun.
An enzyme, -acetylserine(thiol)lyase (OASTL), also known as -acetylserine sulfhydrylase or cysteine synthase (CSase), catalyses the incorporation of sulfide into -acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from , (), but a recombinant SoCSaseLP produced in did not show OASTL activity. The exon-intron structure of the gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione -transferase (GST)-SoCSaseLP fusion protein was expressed in together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.
一种名为β-乙酰丝氨酸（硫醇）裂解酶（OASTL）的酶，也被称为β-乙酰丝氨酸巯基酶或半胱氨酸合酶（CSase），催化将硫化物掺入β-乙酰丝氨酸并产生半胱氨酸。我们之前从菠菜（Spinacia oleracea）中鉴定出一个编码类OASTL蛋白的cDNA，但在大肠杆菌（Escherichia coli）中产生的重组SoCSaseLP并未表现出OASTL活性。菠菜基因的外显子-内含子结构与其他菠菜OASTL基因具有保守结构。SoCSaseLP和一个菠菜同源蛋白KMT13462在OASTL家族的系统发育树中构成一个独特的分支。有趣的是，当该基因在烟草植株中表达时，烟草叶片中的总OASTL活性降低。总OASTL活性的这种降低很可能是由SoCSaseLP对胞质OASTL的干扰所致。为了研究SoCSaseLP与菠菜胞质OASTL同工型SoCSaseA之间可能的相互作用，进行了一项下拉实验。重组谷胱甘肽-S-转移酶（GST）-SoCSaseLP融合蛋白与组氨酸标签化的SoCSaseA蛋白一起在大肠杆菌中表达，并且蛋白质提取物进行谷胱甘肽亲和层析。组氨酸标签化的SoCSaseA与GST-SoCSaseLP融合蛋白共纯化，表明SoCSaseLP与SoCSaseA结合。与这种相互作用一致，与单独纯化的SoCSaseA的活性相比，共纯化的SoCSaseA的OASTL活性降低。这些结果有力地表明，SoCSaseLP通过直接相互作用对其他胞质OASTL家族成员的活性进行负调控。
