Structure of the New Motifs for Nucleotide-binding

在这项名为“核苷酸结合新模体的结构”的联合研究项目中，我们研究了人NADH-细胞色素b5还原酶(B5R)、玉米硝酸盐还原酶(NR)和大鼠NADPH-细胞色素P450还原酶的核苷酸结合模体的结构。在b5R中，C-末端的β链富含疏水氨基酸残基，这些残基从晶体结构上对稳定核苷酸的疏水环境具有重要作用，并有助于与酶结合。即使这些疏水氨基酸残基中的一个与丙氨酸交换，酶的活性也不会受到影响，但当它被诱变删除时，酶的活性会受到很大的影响。这些事实表明，C末端的疏水性对于稳定核苷酸FAD的结合是重要的。为了了解FAD结合域和细胞色素b结构域与NR cDNA的融合蛋白NR中的电子传递，融合蛋白在酵母中得到表达，并通过Blue-Sepharose亲和层析进行纯化。P450R含有FMN和FAD两个核苷酸，还有一个很长的插入序列(120个残基)是N-末端结构域。为了阐明FMN和FAD的结合与长插入序列之间的关系，我们现在正在制备一个嵌合体蛋白，将P450还原酶的N末端结构域与b5R的N末端结构域互换。

In this Joint Research Program entitled "Structure of the New Motifs for Nucleotide-binding", we studied on the structures of the nucleotide-binding motifs of human NADH-cytopchrome b5 reductase (b5R), corn nitrate reductase (NR), and rat NADPH-cyto chrome P450 reductases.In b5R,the C-terminal beta-strand is rich in hydrophobic amino acid residues, and these residues are shown to be important from the crystal structure to stabilize the hydrohobic environment of nucleotide, FAD to bind the enzyme. Even if one of these hydrophobic amino acid residues was exchanged with Alanine, the enzyme activity was not impaired, but when it was deleted by mutagenesis, the enzyme actrivity was highly impaired. These facts indicate that those hydrophobicity around the C-terminus is important to stabilize the binding of nucleotide, FAD.To understand the electron transfer in NR,a fusion protein of the FAD-binding domain and cytochrom b domain with NR cDNA.The fusion protein was expressed in yeast Pichia, and was purified by using an affinity chromatography on a Blue-Sepharose. The fusin protein is now applying to crystalize.P450R contains two nucleotides, FMN and FAD,and also has a long insertion sequence (120 residues) is the N-terminal domain. To clarify the relationship between the binding of FMN and FAD,and the long insertion sequence, we are now preparing a chimera protein exchanging the N-terminal domain of P450 reductase with the N-terminal domain of b5R.