Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli
Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli
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DOI：
10.1093/jb/mvh111
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发表时间：
2004-08-01
影响因子：
2.7
通讯作者：
Noguchi, M
中科院分区：
文献类型：
--
作者：
Omata, Y;Sakamoto, H;Noguchi, M
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（Urogen）III合酶的协同作用产生的。虽然尿苷原III合酶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，尿苷原III合酶还有待研究。本文报道了人尿原Ⅲ合酶的大肠杆菌表达系统和纯化方法。裂解物中的酶是不稳定的，但我们发现甘油防止裂解物中的活性损失。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。对5，5 ′-二硫代双（2-硝基苯甲酸）可接近的Cys残基数目与热灭活期间剩余活性之间的关系的检查表明，特定的Cys残基参与活性损失。从人尿苷原III合酶的晶体结构[马修斯（Mathews）等人（2001）欧洲分子生物学组织杂志（EMBO J.）20，5832-5839]，认为该Cys残基是Cys 73，其深埋在酶内，表明人尿苷原III合酶的Cys 73在酶活性中起重要作用。
The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. 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 be tween the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.