ポーター トッド D. ケンタッキー大学, 薬学部, 助教授
キャンベル ウイルバー ミシガン工科大学, 生化学, 教授
CAMPBELL Wilbur h Michigan Technological University, Professor
PORTER Todd d University of Kentucky, Associate Professor
キャンベル ウイルバーH ミシガン工科大学, 生化学, 教授
|Budget Amount *help
¥3,900,000 (Direct Cost : ¥3,900,000)
Fiscal Year 1995 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1994 : ¥2,000,000 (Direct Cost : ¥2,000,000)
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.