日本落叶松谷胱苷肽还原酶的生化特性研究

本研究从日本落叶松中克隆到一个谷胱苷肽还原酶(GR)基因,命名为LaGR。该基因编码563个氨基酸组成的蛋白质,预测分子量为61.06 kDa。表达模式分析发现LaGR基因在芽、成熟针叶、茎韧皮部和根韧皮部均表达,是组成型表达基因。蛋白质亚细胞定位研究发现LaGR蛋白定位在叶绿体。在大肠杆菌中表达并纯化了LaGR重组蛋白。酶学性质分析发现,LaGR蛋白对底物GSSG和NADPH具有较高的催化活性和亲和力,是热稳定蛋白,而且最适pH值范围在7.0 9.0之间。Cd²⁺、Pb²⁺和Cu²⁺等重金属离子对LaGR蛋白的催化活性具有明显的抑制作用。将LaGR蛋白的第528位His突变为Gln后,其突变蛋白的催化活性显著降低,而且与野生型LaGR蛋白相比,突变蛋白的动力学常数、最适pH值范围和热稳定性均发生了显著变化,预示着第528位的His在LaGR的催化特性和蛋白结构稳定性方面发挥着重要作用。

Biochemical Characterization of Larix kaempferi Glutathione Reductase

Glutathione reductases (GRs) are key enzymes in plant glutathione-ascorbate cycle, and play important roles in plant oxidative stress tolerance. In this study, a GR gene (LaGR) was cloned from Larix kaempferi. This gene encodes protein of 563 amino acid residues, with calculated molecular masses of 61.06 kDa. RT-PCR reveals that LaGR is a constitutive expression gene, which is expressed in all tissues detected, including bud, mature needle, phloem of stem and root. The chloroplast localization of LaGR was confirmed by transient expression in Arabidopsis protoplast. LaGR was overexpressed in Escherichia coli, and purified by Ni-affinity chromatography. The purified LaGR proteins showed high catalytic activities and affinities towards substrates GSSG and NADPH. LaGR protein is a thermostable protein, and has optimal pH ranging from 7.0 to 9.0. Heavy metal ions (Cd²⁺, Pb²⁺ and Cu²⁺) could inhibit the LaGR's activities. When the His528 of LaGR protein was replaced by Gln, the mutant proteins showed decreased enzymatic activities and increased affinity towards substrates GSSG and NADPH. Moreover, the mutant protein showed less thermostability than wild-type protein. Thus, His528 of LaGR protein might contribute to the enzyme's catalytic activity and structural stability.