Structures and functions of metalloflavoenzymes which produce free radicals

产生自由基的金属黄素酶的结构和功能

• 批准号: 09480167
• 负责人: NISHINO Takeshi
• 金额: $ 7.81万
• 依托单位: NIPPON MEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-09480167/
• 关键词: reactive oxygen species; xanthine dehydrogenase; xanthine oxidase; niric oxide synthase; metallo-protein; non-heme iron; peroxiredoxin; heme binding protein; NO合成酵素; 金属フラビン酵素; セロビオース脱水素酵素; 電子スピン共鳴; X線結晶構造; フリーラジカル; EPR; nNOS; Xanthine Oxid

Xanthine dehydrogenase/oxidase and nitric acid synthetase are complex flavoprotein that produce free radicals. In this study, we presented the crystal structure of the dimeric (Mr 290,000) bovine milk XDH at 2.1 Å resolution and XO at 2.5 Å resolution and describe the major changes that occur upon the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20 kDa domain containing two iron sulfur centers, a central 40 kDa FAD domain, and a C-terminal 85 kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gin-423-Lys-433). This movement partially blocks access of the NAD substrate to the FAD cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme. We have also constructed several mutant enzyme of rat liver XDH and determined the character to confirm the responsible residues for conversion.We have expressed mouse neuronal nitic acid synthase (NOS) as well as the iso-form of natural mutant and determined characters of the enzymes by several physicochemical methods. We have also studied some other related enzymes such as heme containing flavo-protein, cellobiose dehydrogenase, and heme binding protein, HBP23 has peroxidase activity.

黄嘌呤脱氢酶/氧化酶和硝酸合成酶是产生自由基的复杂黄素蛋白。在这项研究中，我们展示了二聚体 (Mr 290,000) 牛乳 XDH 的晶体结构（分辨率为 2.1 Å）和 XO 的晶体结构（分辨率为 2.5 Å），并描述了主要变化。 XDH 蛋白水解转化为 XO 形式时发生的每个分子均由 N 端 20 组成。 kDa 结构域包含两个铁硫中心、一个中心 40 kDa FAD 结构域和一个 C 端 85 kDa 钼蝶呤结合结构域，其中四个氧化还原中心以几乎线性的方式排列。XDH 表面暴露环的裂解导致主要结构重排。靠近黄素环的另一个环 (Gin-423-Lys-433) 这种运动部分阻止了 NAD 底物与 FAD 的接触。我们还构建了大鼠肝脏 XDH 的几种突变酶，并确定了其特征以确认负责转换的残基。我们已经表达了小鼠神经元硝酸合酶（NOS）以及天然突变体的异构体，并通过多种物理化学方法确定了酶的特性，我们还研究了一些其他相关酶，例如含有黄素蛋白的血红素，纤维二糖脱氢酶和血红素结合蛋白HBP23具有过氧化物酶活性。

项目成果

T.Nishino,Y.Kashima,K.Okamoto,T.Iwasaki,T.Nishino: "Favins and Flavoproteins,Calgary Press," K.Stevenson,CH.Williams and V.Massey eds., 843-846 (1997)

T.Nishino、Y.Kashima、K.Okamoto、T.Iwasaki、T.Nishino：“Favins 和黄素蛋白，卡尔加里出版社”，K.Stevenson、CH.Williams 和 V.Massey 编辑，843-846 (1997)

H.Hori,T.Iwasaki,Y.Kurahashi, and T.Nishino: "Calcium Dependent Inactivation of Neuronal Nitric Oxide Synthase: Evidence for the Existence of Stabilization/Activation Factor."Biochem. Biophys. Res. Commun.. 234. 476-480 (1997)

H.Hori、T.Iwasaki、Y.Kurahashi 和 T.Nishino：“神经元一氧化氮合酶的钙依赖性失活：稳定/激活因子存在的证据。”生物化学。

K.Okamoto,T.Nishino: "Favins and Flavoproteins,Calgary Press," K.Stevenson,CH.Williams and V.Massey eds., 839-842 (1997)

K.Okamoto、T.Nishino：“Favins 和黄素蛋白，卡尔加里出版社”，K.Stevenson、CH.Williams 和 V.Massey 编辑，839-842 (1997)

K.Igarashi,M.Verhagen,M.Samejima,M.Schulein,K.L.Eriksson and T.Nishino: "Cellobiose dehydrogenase from the fungi Phanerochaete chrysoporium and Humicola insolens; A flavoprotein from Humicola insolens contains 6-hydroxy-FAD as the dominant active cofactor

K.Igarashi、M.Verhagen、M.Samejima、M.Schulein、K.L.Eriksson 和 T.Nishino：“来自真菌 Phanerochaete chrysoporium 和 Humicola insolens 的纤维二糖脱氢酶；来自 Humicola insolens 的黄素蛋白含有 6-羟基-FAD 作为主要活性物质

Y.Niimura,K.Ohnishi,et al.,T.Nishino: "Favins and Flavoproteins,Calgary Press," K.Stevenson,CH.Williams and V.Massey eds., 741-750 (1997)

Y.Niimura、K.Ohnishi 等人、T.Nishino：“Favins 和黄素蛋白，卡尔加里出版社”，K.Stevenson、CH.Williams 和 V.Massey 编辑，741-750 (1997)