Investigation of the mechanism of D-ring inversion in porphyrin biosynthesis

卟啉生物合成中D环反转机制的研究

• 批准号: 14580658
• 负责人: OMATA Yoshiaki
• 金额: $ 1.6万
• 依托单位: Kurume University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580658/
• 关键词: prophyrin; uroporphyrinogen; uroporphyrinogen III syntase; congenital erythropoietic porphyria; hydroxymethylbilane; hydroxymethylbilane synthase

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen III synthase(UROS). Although the role of UROS is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of UROS have yet to be studied. An expression system in E.coli and a purification procedure for human UROS were established in this project. The enzyme in the lysate was unstable, but glycerol is able to prevent the activity loss in the lysate for at least a few days after lysis. The enzyme therefore was purified in the presence of glycerol, the entire procedure being completed within 48 hr after lysis with the aid of an HPLC system. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of accessible Cys residues and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human UROS, this Cys residue was considered to be Cys73, which is deeply buried inside the enzyme. A mutation of Cys73 to Arg is most common in congenital erythropoietic porphyria(CEP), in which UROS is deficient, having been found in the allele of 33% of CEP patients. These findings suggest that Cys73 of human UROS has an important role in catalysis. From the results of chemical modifications that indicate the role of several basic residues, Lys, Arg and His residues around putative active site were mutated. The activity and kinetic parameters of mutated enzymes suppose that substrate is bound between two opposite residues and surrounded by the other residues.

生理性卟啉的侧链不对称性是由羟甲基胆烷合酶和尿卟啉原III合酶（UROS）的协同作用产生的。虽然UROS的作用是必不可少的卟啉生物合成的化学，许多方面，结构以及机械，UROS还有待研究。本课题建立了人UROS的原核表达系统和纯化方法。裂解物中的酶不稳定，但甘油能够在裂解后至少几天内防止裂解物中的活性损失。因此，在甘油存在下纯化酶，整个过程在裂解后48小时内借助HPLC系统完成。纯化的酶显示出显着的热稳定性，特别是当保持在磷酸盐缓冲液中含有DTT或EDTA，表明酶的活性可能取决于其氧化态。在热失活过程中可接近的Cys残基的数量和剩余的活性之间的关系的检查表明，一个特定的Cys残基参与活性损失。根据人UROS的晶体结构，该Cys残基被认为是Cys73，其深埋在酶内。Cys73突变为Arg在先天性红细胞生成性卟啉症（CEP）中最常见，其中UROS缺陷，在33%的CEP患者的等位基因中发现。这些发现表明人类UROS的Cys 73在催化中具有重要作用。从化学修饰的结果表明，几个碱性残基的作用，赖氨酸，精氨酸和组氨酸残基周围的推定的活性位点进行了突变。突变酶的活性和动力学参数假设底物结合在两个相对的残基之间，并被其他残基包围。

项目成果

Crystal structure of rat apo-hemeoxygenase-1(HO-1) : Mechanism of heme binding in HO-1 inferred from strutural comparison of the apo and heme

大鼠 apo-hemeoxygenase-1(HO-1) 的晶体结构：从 apo 和血红素的结构比较推断 HO-1 中血红素结合的机制

• 发表时间: 2002
• 期刊: Biochemistry 41
• 作者: Masakazu Sugishima

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

• DOI: 10.1093/jb/mvh111
• 发表时间: 2004-08-01
• 期刊: JOURNAL OF BIOCHEMISTRY
• 影响因子: 2.7
• 作者: Omata, Y;Sakamoto, H;Noguchi, M
• 通讯作者: Noguchi, M

Crystal structure of rat hteme oxygenase-1 in complex with heme bound to azide : Implication for regiospecific hydroxylation of heme at the

大鼠 hteme 加氧酶-1 与与叠氮化物结合的血红素复合物的晶体结构：对血红素在

• 发表时间: 2002
• 期刊: The Journal of Biological Chemistry 277
• 作者: Masakazu Sugishima

The reactivity of α-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite.

α-羟基血红素和维多血红素与血红素加氧酶-1 结合，生成双氧和连二亚硫酸钠。

• 发表时间: 2002
• 期刊: Eur.J.Biochem. 269-21
• 作者: Hiroshi Sakamoto;Yoshiaki Omata;Shunsuke Hayashi;Saori Harada;Graham Palmer;Masato Noguchi
• 通讯作者: Masato Noguchi

Hiroshi Sakamoto: "The reactivity of a-hydroxyhaem and verdohaem bound to haemoxygenase-1 to dioxygen and sodium dithionite."Europian Journal of Biochemistry. 269. 5231-5239 (2002)

Hiroshi Sakamoto：“α-羟基血红素和维多血红素与血加氧酶 1 结合，生成双氧和连二亚硫酸钠的反应性。”《欧洲生物化学杂志》。