Ye Hong, Abdel-Ghany Salah E, Anderson Timothy D, Pilon-Smits Elizabeth A H, Pilon Marinus
Biology Department, Colorado State University, Fort Collins, Colorado 80523.
J Biol Chem. 2006 Mar 31;281(13):8958-69. doi: 10.1074/jbc.M512737200. Epub 2006 Feb 2.
CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.
CpNifS是拟南芥叶绿体中为铁硫簇生物合成提供硫所需的一种半胱氨酸脱硫酶，属于一类内源性半胱氨酸脱硫酶活性较低的NifS样酶。其细菌同源物SufS受SufE刺激。在这里，我们对拟南芥叶绿体蛋白CpSufE进行了表征，它具有一个N端的SufE样结构域和一个高等植物特有的C端BolA样结构域。绿色荧光蛋白定位和免疫印迹实验表明，CpSufE定位于叶绿体基质。与CpNifS一样，CpSufE在所有主要组织中均有表达，在绿色部分表达较高。其表达受光调控且在mRNA水平上受到调节。添加纯化的重组CpSufE可使CpNifS的半胱氨酸脱硫酶活性的Vmax增加40多倍，并使对半胱氨酸的KM从0.1毫米降至0.043毫米。相比之下，添加CpSufE会降低CpNifS对硒代半胱氨酸的亲和力，表现为KM从2.9毫米增加到4.17毫米，并使硒代半胱氨酸裂解酶活性的Vmax降低30%。凝胶过滤、非变性聚丙烯酰胺凝胶电泳和亲和色谱实验表明，CpSufE与CpNifS形成动态复合物。CpSufE中的单个半胱氨酸突变为丝氨酸的突变体在刺激CpNifS方面没有活性，尽管它确实与野生型CpSufE竞争。CpNifS-CpSufE复合物对脱辅基铁氧化还原蛋白的铁硫簇重构活性比单独的CpNifS高20倍。我们得出结论，CpNifS和CpSufE共同形成叶绿体中铁硫簇形成所需的半胱氨酸脱硫酶。
