Roles and specific functions of homologous enzymes involved in biogenesis of active-form sulfur and active-form selenium

参与活性硫和活性硒生物发生的同源酶的作用和特定功能

• 批准号: 11480179
• 负责人: ESAKI Nobuyoshi
• 金额: $ 9.22万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480179/
• 关键词: selenocysteine lyase; cysteine desulfurase; selenophosphate; selenium; iron-sulfur cluster; sulfur

Mechanism and physiological role of cysteine desulfurase which decomposes cysteine into alanine and sulfur and its homolog, selenocysteine lyase, were studied. Two cysteine desulfurases from a cyanobacterium were used to generate an iron-sulfur cluster in an iron-sulfur protein. The enzymes efficiently converted apo-ferredoxin into [2Fe-2S] ferredoxin by using L-cysteine as a sulfur source. Selenocysteine lyase from Escherichia coli was employed to synthesize selenophosphate in vitro. Selenophosphate was efficiently produced in the presence of selenophosphate synthetase, selenocysteine, and ATP.Three gene-disrupted E.coli strains, in which a gene for each cysteine desulfurase homolog was disrupted, were constructed to investigate role of each enzyme. Growth rate of the iscS-disruptant was significantly decreased on a LB plate medium. The iscS-disruptant was transformed with plasmids containing genes for CSD, CsdB, IscS and their mutant enzymes, and growth-rates of the transformatnts were investigated on various solid media. Introduction of csdB into the iscS-disruptant recover the growth-rate of the strain to the level as the wild-type strain. The result suggests that the over-produced CsdB can be a back-up of IscS in vivo. On the other hand, expression of CSD did not complement the growth of the iscS-disruptant. Thus, the in vivo functions of CSD and CsdB are different from each other, although they show about 50% amino acid identity.

研究了半胱氨酸脱硫酶及其同系物硒代半胱氨酸裂解酶的作用机制和生理功能。两个半胱氨酸脱硫酶从蓝藻被用来产生铁硫蛋白中的铁硫簇。以L-半胱氨酸为硫源，该酶能有效地将脱辅基铁氧还蛋白转化为[2Fe-2S]铁氧还蛋白。利用大肠杆菌硒代半胱氨酸裂解酶体外合成硒代磷酸盐。硒磷酸合成酶，硒代半胱氨酸，和ATP.Three基因破坏的大肠杆菌菌株，其中每个半胱氨酸脱硫酶同系物的基因被破坏的存在下，有效地产生，构建调查的作用，每个酶。在LB平板培养基上，iscS-破坏剂的生长速率显著降低。用含有CSD、CsdB、IscS及其突变酶基因的质粒转化iscS-破坏物，并研究了转化物在各种固体培养基上的生长速率。将csdB引入到iscS-破坏剂中，使菌株的生长速率恢复到野生型菌株的水平。结果表明，过量产生的CsdB可以作为体内IscS的备份。另一方面，CSD的表达不补充iscS-破坏物的生长。因此，CSD和CsdB的体内功能彼此不同，尽管它们显示出约50%的氨基酸同一性。

项目成果

Hisaaki Mihara et al.: "cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase. Candidate for selenium delivery protein in selenoprotein synthesis."J.Biol.Chem.. 275. 6195-6200 (2000)

Hisaaki Mihara 等人：“小鼠肝脏硒代半胱氨酸裂解酶的 cDNA 克隆、纯化和表征。硒蛋白合成中硒传递蛋白的候选者。”J.Biol.Chem.. 275. 6195-6200 (2000)

Gerard Lacourciere et al.: "Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate"The Journal of Biological Chemistry. 275(31). 23796-23773 (2000)

Gerard Lacourciere 等人：“大肠杆菌 NifS 样蛋白在硒磷酸盐生物合成途径中提供硒”《生物化学杂志》。

Hisaaki Mihara et al.: "Kinetic and mutational studies of three NifS homologs from Escherichia coli : Mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions."J.Biochem.. 127. 559-567 (2000)

Hisaaki Mihara 等人：“来自大肠杆菌的三种 NifS 同源物的动力学和突变研究：L-半胱氨酸脱硫酶和 L-硒代半胱氨酸裂解酶反应之间的机制差异。”J.Biochem.. 127. 559-567 (2000)

Hisaaki Mihara et al.: "A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase. Gene cloning, purification, characterization and preliminary x-ray crystallographic studies."J.Biol.Chem.. 274. 14768-14772 (1999)

Hisaaki Mihara 等人：“类似 nifS 的基因 csdB 编码哺乳动物硒代半胱氨酸裂解酶的大肠杆菌对应物。基因克隆、纯化、表征和初步 X 射线晶体学研究。”J.Biol.Chem.. 274. 14768

Hisaaki Mihara et al.: "A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase"The Journal of Biological Chemistry. 274(21). 14768-14772 (1999)

Hisaaki Mihara 等人：“nifS 样基因 csdB，编码哺乳动物硒代半胱氨酸裂解酶的大肠杆菌对应物”《生物化学杂志》。