Lancelin J M, Guilhaudis L, Krimm I, Blackledge M J, Marion D, Jacquot J P
Laboratoire de RMN Biomoléculaire associé au CNRS, Université Claude Bernard-Lyon 1 and Ecole Supérieure de Chimie, Physique et Electronique de Lyon, Villeurbanne, France.
Proteins. 2000 Nov 15;41(3):334-49. doi: 10.1002/1097-0134(20001115)41:3<334::aid-prot60>3.3.co;2-d.
Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.
来自莱茵衣藻的叶绿体硫氧还蛋白m在光还原铁氧还蛋白和铁氧还蛋白依赖性铁氧还蛋白-硫氧还蛋白还原酶存在的情况下，在体外和体内都能非常有效地被还原。一旦被还原，硫氧还蛋白m就有能力快速激活NADP苹果酸脱氢酶（EC 1.1.1.82），这是一种参与C4植物中能量依赖性二氧化碳同化的调节酶。这种激活是硫氧还蛋白m还原位于NADP苹果酸脱氢酶N端和C端的两个二硫键的结果。利用核磁共振技术解析了硫氧还蛋白m的分子结构，并与其他已知的硫氧还蛋白进行了比较。硫氧还蛋白m属于原核型硫氧还蛋白，与真核型硫氧还蛋白不同，真核型硫氧还蛋白在植物中也由h（胞质）型和f（叶绿体）型硫氧还蛋白代表。利用（15）N弛豫数据评估了该分子的动力学，发现其与计算出的核磁共振系综中发现的无序区域相关性良好。所得结果为解释叶绿体NADP-苹果酸脱氢酶激活的硫氧还蛋白依赖性提供了新的基础。还根据最近的新认识，特别是鉴于对氧化还原活性位点的两个半胱氨酸施加的双重酸碱催化作用，讨论了硫氧还蛋白活性位点发生的具体催化机制。有人提出，氧化还原活性位点的两个半胱氨酸可能通过不变的疏水氨基酸的特定堆积相互隔离，免受溶剂攻击。
