Structure of the New Motifs for Nucleotide-binding

在“核苷酸结合新基序的结构”联合研究项目中，我们研究了人类nadh -细胞色素b5还原酶（b5R）、玉米硝酸盐还原酶（NR）和大鼠nadph -细胞铬P450还原酶的核苷酸结合基序的结构。在b5R中，c端β链富含疏水氨基酸残基，这些残基从晶体结构上被证明是稳定核苷酸、FAD结合酶的疏水环境的重要因素。即使这些疏水氨基酸残基中的一个与丙氨酸交换，酶活性也不会受到损害，但当它被诱变删除时，酶活性受到严重损害。这些事实表明，c端周围的疏水性对于稳定核苷酸、FAD的结合是重要的。为了了解NR中的电子转移，研究了一种fad结合域和细胞染色质b域与NR cDNA的融合蛋白。融合蛋白在酵母毕赤酵母中表达，并通过Blue-Sepharose亲和层析纯化。熔融蛋白现在开始结晶。P450R含有FMN和FAD两个核苷酸，在n端结构域也有一个很长的插入序列（120个残基）。为了明确FMN与FAD结合与长插入序列之间的关系，我们正在制备一种将P450还原酶的n端结构域与b5R的n端结构域交换的嵌合体蛋白。

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.