玉米过氧化物还原蛋白BAS1的原核表达及其功能研究

Prokaryotic Expression and Functional Analysis of Maize BAS1(2-Cys peroxiredoxinA)

植物过氧化物还原蛋白BAS1是巯基依赖的过氧化物酶,通过催化的Cys残基还原过氧化氢,依赖NADPH的叶绿体硫氧还蛋白还原酶保持BAS1的还原态。玉米含有两种BAS1:2-CysPrxA和2-CysPrxB。利用RT-PCR方法从玉米幼叶中克隆了编码成熟2-CysPrxA的基因,并将蛋白Cys34残基突变成Ser34。SDS-PAGE显示纯化的野生型和突变体蛋白为一条主带,分子量约为23kDa;体外蛋白结合实验表明纯化的叶绿体硫氧还蛋白还原酶通过分子间二硫键结合纯化的2-CysPrxA的C34S突变体,非还原SDS-PAGE显示纯化的野生型2-CysPrxA含有分子间二硫键组成的二体,而纯化的C34S突变体呈现单体,巯基专一性标记化合物AMS修饰及活性分析表明纯化的BAS1还原态是催化还原过氧化氢所所必须的,它由硫氧还蛋白还原酶及其辅酶NADPH所催化。

The plant 2-Cys peroxiredoxin BAS1 is a thiol-dependent peroxidase capable of reducing hydrogen peroxide through the reactive catalytic cysteine,which is regenerated by NADPH dependent thioredoxin reductase from chloroplast（NTRC）.Maize contains two BAS1,2-Cys peroxiredoxin A（2-Cys PrxA）and 2-Cys peroxiredoxin B（2-Cys PrxB）.The BAS1 gene encoding the mature 2-Cys PrxA from maize leaves was amplified by RT-PCR,and Cys34 was mutated into Ser34 in the BAS1 by site-directed mutagenesis.The SDS-PAGE analysis displayed that the subunit of the purified BAS1 wild type and mutant C34S has the apparent molecular mass about 23kDa.The protein binding assay showed the purified BAS1 mutant C34S is associated with the purified NTRC through the intermolecular disulfide bridge.Non-reducing SDS-PAGE analysis proved the purified BAS1 wild type formed the dimer through the intermolecular disulfide bridge,whereas the purified BAS1 mutant C34S formed the monomer.The results of modification BAS1 with thiol-reactive labeling reagent AMS and the activity analysis identified that the reducing BAS1 is essential for the catalysis of the reduction of hydrogen peroxide,which is kept by NTRC and its cofactor NADPH.