Structure of the New Motifs for Nucleotide-binding

核苷酸结合新基序的结构

• 批准号: 06044186
• 负责人: YUBISUI Toshitsugu
• 金额: $ 2.5万
• 依托单位: Kochi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044186/
• 关键词: Nucleotide-binding motif; Cytochrome b5 reductase; Nitrate reductase; Crystal structure; Mutagenesis; Cytochrome P450 reductase; ヌクレオチド; モチーフ; NADH-シトクロムb5還元酵素; ミノ酸置換

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.

在题为“新的核苷酸结合基序的结构”的联合研究计划中，我们研究了人NADH-细胞Chrome b5还原酶（b5 R）、玉米硝酸还原酶（NR）和大鼠NADPH-细胞Chrome P450还原酶的核苷酸结合基序的结构。在b5 R中，C-末端β-链富含疏水性氨基酸残基，从晶体结构上看，这些残基对稳定核苷酸的疏水环境、FAD结合酶具有重要作用。即使这些疏水性氨基酸残基之一与丙氨酸交换，酶活性也不受损，但是当通过诱变将其缺失时，酶活性高度受损。为了解NR中的电子传递，将FAD结合结构域和细胞色素B结构域与NR cDNA融合，在毕赤酵母中表达融合蛋白，并通过Blue-Sepharose亲和层析纯化。P450 R含有FMN和FAD两个核苷酸，N端有一个长的插入序列（120个残基）。为了阐明FMN和FAD的结合与长插入序列之间的关系，我们现在制备将P450还原酶的N-末端结构域与b5 R的N-末端结构域交换的嵌合蛋白。

项目成果

Guoguang Lu: "Structural Studies on Corn Nitrate Reductase : Refined Structure of the Cytochrome b Reductase at 2.5A,its ADP Complex" J.Mol.Biol.248. 931-948 (1995)

陆国光：“玉米硝酸还原酶的结构研究：2.5A 细胞色素 b 还原酶及其 ADP 复合物的精细结构”J.Mol.Biol.248。

Shirabe,K.: "An in-frame deletion of codon 298 of the NADH-cytochrome b5 reductase gene results in hereditary methemoglobinemia type II" J.Biol.Chem.269. 5952-5957 (1994)

Shirabe,K.：“NADH-细胞色素 b5 还原酶基因密码子 298 的框内删除会导致 II 型遗传性高铁血红蛋白血症”J.Biol.Chem.269。

Lu,G.: "Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5A resolution" Structure. 2. 809-812 (1994)

Lu,G.：“2.5A 分辨率下含有 FAD 的玉米硝酸还原酶片段的晶体结构” 结构。

Terry E.Meyer: "Transient kinetics of Intracomplex Electron Transfer in the Human Cytochrome b5 Reductase-Cytochrome b5 System" Arch.Biochem.Biopys.318. 457-464 (1995)

Terry E.Meyer：“人细胞色素 b5 还原酶-细胞色素 b5 系统中复合物内电子转移的瞬时动力学”Arch.Biochem.Biopys.318。

Guoguang Lu: "Structural Studies on Corn Nitrate Reductase Refined Structure of the Cytochrome b Reductase Fragment at 2.5A,its ADP Compl" J.Mol.Biol.248. 931-948 (1995)

陆国光：“玉米硝酸还原酶2.5A细胞色素b还原酶片段及其ADP复合物精制结构的结构研究”J.Mol.Biol.248。