Sundaram Sabarinath, Rathinasabapathi Bala, Ma Lena Q, Rosen Barry P
Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, Gainesville, FL 32611-0690, USA.
J Biol Chem. 2008 Mar 7;283(10):6095-101. doi: 10.1074/jbc.M704149200. Epub 2007 Dec 23.
To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.
为阐明砷超富集蕨类植物蜈蚣草对砷的抗性机制，基于一个cDNA能增强大肠杆菌对砷的抗性，从其叶片表达cDNA文库中分离出一个谷氧还蛋白（Grx）Pv5-6的cDNA。Pv5-6推导的氨基酸序列与拟南芥叶绿体Grx具有高度同源性，并含有两个CXXS假定催化基序。纯化的重组Pv5-6表现出谷氧还蛋白活性，10 mM砷酸盐可使其活性提高1.6倍。将半胱氨酸（Cys）（67）定点突变为丙氨酸（Ala）（67）导致GRX活性和抗砷性丧失。PvGrx5在缺失ars操纵子砷抗性基因的大肠杆菌突变体（菌株AW3110）、缺失ArsC砷酸盐还原酶基因的菌株（菌株WC3110）以及ars操纵子缺失且GlpF水甘油孔蛋白基因被破坏的菌株（菌株OSBR1）中表达。PvGrx5的表达提高了AW3110和WC3110菌株对砷的耐受性，但在OSBR1中没有，这表明PvGrx5在细胞抗砷中发挥作用，独立于ars操纵子基因，但依赖于GlpF。与载体对照相比，在含有2.5 mM砷酸盐的培养基中培养时，表达PvGrx5的AW3110细胞中的亚砷酸盐水平显著降低。我们的结果与PvGrx5通过直接或间接调节水甘油孔蛋白来调节细胞内亚砷酸盐水平的作用一致。据我们所知，PvGrx5是第一个涉及砷代谢的植物Grx。
