de Lamotte-Guery F, Miginiac-Maslow M, Decottignies P, Stein M, Minard P, Jacquot J P
Laboratoire de Physiologie Végétale Moléculaire, Université Paris Sud, Orsay, France.
Eur J Biochem. 1991 Mar 14;196(2):287-94. doi: 10.1111/j.1432-1033.1991.tb15816.x.
A new over-expression system has been set up for Escherichia coli thioredoxin, yielding 55 mg purified protein/10 g fresh cells. This system has been used to produce thioredoxin modified by site-directed mutagenesis. Taking advantage of the structural and enzymatic similarity between E. coli and spinach m-type thioredoxin, Asp61 of E. coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes. The modification did not significantly alter the structure of the protein. Neither the rate of reduction of insulin and 5,5'-dithio-bis(2-nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH-dependent thioredoxin reductase, have been modified. The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin-dependent thioredoxin reductase in a light-activation reconstituted chloroplast system. The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose-1,6-bisphosphatase activation by the dithiothreitol-reduced thioredoxin, and to an increase in both spinach fructose-1,6-bisphosphatase and corn NADP-dependent malate dehydrogenase activities in the light-activation system. This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose-1,6-bisphosphatase and ferredoxin-dependent thioredoxin reductase.
已建立一种新的大肠杆菌硫氧还蛋白过表达系统，每10克新鲜细胞可产生55毫克纯化蛋白。该系统已用于生产通过定点诱变修饰的硫氧还蛋白。利用大肠杆菌和菠菜m型硫氧还蛋白之间的结构和酶学相似性，将大肠杆菌硫氧还蛋白的Asp61突变为Asn，以研究抑制带电荷残基对硫氧还蛋白与靶酶相互作用的影响。这种修饰并未显著改变蛋白质的结构。还原型硫氧还蛋白对胰岛素和5,5'-二硫代双(2-硝基苯甲酸)的还原速率，以及NADPH依赖性硫氧还蛋白还原酶的还原作用均未改变。在光激活重建叶绿体系统中，用二硫苏糖醇还原的硫氧还蛋白和用铁氧还蛋白依赖性硫氧还蛋白还原酶还原的硫氧还蛋白对叶绿体酶激活的主要影响被观察到。用中性的Asn取代带负电荷的Asp61，导致二硫苏糖醇还原的硫氧还蛋白激活菠菜果糖-1,6-二磷酸酶的效率增加，并且在光激活系统中菠菜果糖-1,6-二磷酸酶和玉米NADP依赖性苹果酸脱氢酶的活性均增加。这表明负电荷的抑制提高了硫氧还蛋白与叶绿体酶如果糖-1,6-二磷酸酶和铁氧还蛋白依赖性硫氧还蛋白还原酶的反应性。
