Gene Technical Approach for Substrate Specificity of Multi-copper Enzymes.

多铜酶底物特异性的基因技术方法。

• 批准号: 09672205
• 负责人: HIROSE Junzo
• 金额: $ 1.73万
• 依托单位: Fukuyama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672205/
• 关键词: Bilirubin Oxidase; Multi-copper Enzyme; Copper Enzyme; Copper Protein; ビリルビンオキシダーゼ; アスコルビン酸オキシダーゼ; ビリルビン・オキシダーゼ; 遺伝子工学; 発現; 基質特異性

We isolated the clone of bilirubin oxidase (BOD) obtained from Trachyderma Tsunodae K-2593 and deduced the amino acid sequence. The sequence has submitted to the DDDBJ/EMBL/GenBank database as AB 006824. On the basis of EPR, visible spectra, and the copper content of BOD, it was shown that of BOD is multi-copper enzyme, which has one type 2 copper, one type 1 copper, and two type 3 copper. The coordination residues of type 1, type2 and type 3 copper was predicted of homology of the amino acid sequence between BOD and ascorbate oxidase obtained from zucchini. Every ligand of type 2 and type 3 copper ions in BOD was shown to be His and was completely consistent with those of zucchini ascorbate oxidase. The ligand of type 1 copper in BOD was Cys, His, His, and Phe. Met which coordinates with type 1 copper in zucchini ascorbate oxidase was replaced by Phe in BOD. The redox potential of type 1 copper of BOD was measured under anaerobic conditions and was 642 mV that was much higher than that zucchini ascorbate oxidase. The higher redox potential of type 1 copper is because the hydrophobicity of Phe around type 1 copper in BOD raises the redox potential. The various anions influenced the visible spectra of type 1 copper in BOD. NィイD23ィエD2ィイD1-ィエD1 and SCNィイD1-ィエD1 gave the increase of the visible absorption spectra, but FィイD1-ィエD1 gave the decrease of it. EPR spectra of the adducts of these anions with BOD clearly indicated that these anion bind to type 2 copper in BOD. These behaviors were interpreted by the disproportination of the electron between type 1 copper and type 2/type 3 cluster. We tried the expression of the gene of BOD in Ecoli, but it was not succeeded.

从沙眼衣原体K-2593中分离得到胆红素氧化酶(BOD)基因，并对其进行氨基酸序列分析。该序列已作为AB 006824提交给生物多样性数据库/欧洲经济数据库/基因数据库。根据EPR、可见光谱和BOD中铜含量的分析，BOD是一种多铜酶，含有1种2型铜、1种1型铜和2种3型铜。根据BOD与西葫芦抗坏血酸氧化酶氨基酸序列的同源性，预测了1、2、3型铜的配位残基。BOD中2型和3型铜离子的每一种配基都是他的，与西葫芦抗坏血酸氧化酶的配基完全一致。BOD中1型铜的配体为Cys、His、His和Phe。西葫芦抗坏血酸氧化酶中与1型铜配位的Met在BOD中被Phe取代。在厌氧条件下测得BOD1型铜的氧化还原电位为642 mV，远高于西葫芦抗坏血酸氧化酶的氧化还原电位。1类铜的氧化还原电位较高是因为BOD中1类铜周围的苯丙氨酸的疏水性提高了氧化还原电位。不同的阴离子对BOD中第一类铜的可见光谱有影响。NィイD23ィエD2ィイD_1-ィエD_1和S_nィイD_1-ィエD_1使可见吸收光谱增加，而F_ィイD_1-ィエD_1使可见吸收光谱减少。这些阴离子与BOD的加合物的EPR谱清楚地表明，这些阴离子与BOD中的第二类铜结合。这些行为可以用第一类铜和第二类/第三类团簇之间的电子分布来解释。我们尝试了BOD基因在大肠杆菌中的表达，但没有成功。

项目成果

Junzo Hirose et al.: "Anions binding to bilirubin oxidase from Trachyderma tsunodae K-2539"Inorganica Chimica Acta. 273. 204-212 (1998)

Junzo Hirose 等人：“与来自 Trachyderma tsunodae K-2539 的胆红素氧化酶结合的阴离子”Inorganica Chimica Acta。

Junzo Hirose, Isao Fjiwara Iio, Shinichi Masunari, Masayoshi Minakami, Luigi Messori and Keitaro Hiromi: "The dependence of the copper dissociation rate constants from human(serum)- and ovo-transferrin on pH and anions."Inorganic Reaction Mechanism.. 1. 1

Junzo Hirose、Isao Fjiwara Iio、Shinichi Masunari、Masayoshi Minakami、Luigi Messori 和 Keitaro Hiromi：“人（血清）和卵转铁蛋白的铜解离速率常数对 pH 和阴离子的依赖性。”无机反应机制.. 1

Junzo Hirose et al.: "Thedependence of the copper dissociation rate constants from human(serum)-and ovo-transferrin on pH and the anions."Inorganic Reation Mechanism. 1. 121-128 (1999)

Junzo Hirose 等人：“人（血清）和卵转铁蛋白的铜解离速率常数对 pH 值和阴离子的依赖性。”无机反应机制。

Katsuhiko Fukasawa, Kayoko M. Fukasawa, Motoko Kana, Shingo Fujii, Junzo Hirose, Mamoru Harada: "Dipeptidyl peptidase is a zinc metallopeptidase"Biochem. J. 329. 275-282 (1998)

Katsuhiko Fukasawa、Kayoko M. Fukasawa、Motoko Kana、Shingo Fujii、Junzo Hirose、Mamoru Harada：“二肽基肽酶是一种锌金属肽酶”Biochem。

Katuhiko Fukasawa et al.: "Dipeptidyl peptidase is a zinc metallopeptidase"Biochem. J.. 329. 275-282 (1998)

Katuhiko Fukasawa 等人：“二肽基肽酶是一种锌金属肽酶”Biochem。