Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli
Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli
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DOI：
10.1093/oxfordjournals.jbchem.a022535
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发表时间：
1999-11-01
影响因子：
2.7
通讯作者：
Nakamura, M
中科院分区：
文献类型：
--
作者：
Takahashi, Y;Nakamura, M
Fe-S 簇是许多蛋白质活性所必需的非血红素-铁辅助因子，通过未知机制整合到其靶蛋白中。在大肠杆菌中，ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3簇（ise基因簇）中的基因应该参与Fe-S簇的组装，因为它与报告铁氧还蛋白（Fd）的共表达显着增加了holoFd的产生[Nakamura，M，Saeki，K.和Takahashi，Y， (1999) J, Biochem, 126, 10-18]，在这项研究中，我们解决了 ise 基因簇编码的蛋白质相对于四个报告 Fd 中 Fe-S 簇组装的功能作用，构建了质粒，其中 ise 基因簇中的 8 个 ORF 通过截短编码区或通过引入包含终止密码子的寡核苷酸接头单独失活，通过将这些质粒与报告 Fd 共表达，我们展示了 Fe-S 簇组装所需的 iscS、iscA、hscA 和 fdx 基因。当共表达质粒中不存在这些基因时，在所检查的任何报告基因 Fd 中都没有实现过量生产。 ORF2和hscB的失活对一些Fd的产生有部分但明显的影响，ORF1的删除与与完整ise基因簇的共表达没有差异。我们还使用 ise 基因簇中的 fdx 基因作为报告基因 Fd 检查了共表达，并鉴定出 iscS、hscB、hscA 和 ORF3 参与了该蛋白质中 [2Fe-2S] 簇的组装。我们提出了一个模型，其中 fdx 基因产物充当 Fe-S 簇组装的中间位点。
Fe-S cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into its target protein by an unknown mechanism. In Escherichia coli, genes in the ORF1-ORF2- iscS-iscU-iscA-hscB-hscA-fdx-ORF3 cluster (the ise gene cluster) should be involved in the assembly of the Fe-S cluster since its coexpression with the reporter ferredoxin (Fd) dramatically increases the production of holoFd [Nakamura, M,, Saeki, K., and Takahashi, Y, (1999) J, Biochem, 126, 10-18], In this study we addressed the functional roles of the proteins encoded by the ise gene cluster with respect to the assembly of Fe-S clusters in four reporter Fds, Plasmids were constructed in which eight ORFs in the ise gene cluster were individually inactivated either by truncating the coding region or by introducing an oligonucleotide linker containing stop codons, By coexpressing these plasmids with reporter Fds, we show the iscS, iscA, hscA, and fdx genes to be required for the assembly of the Fe-S clusters. When these genes were absent from the coexpression plasmid, no overproduction was achieved in any reporter Fds examined. The inactivation of ORF2 and hscB had a partial but appreciable effect on the production of some Fds, Deletion of ORF1 produced no difference from the coexpression with the intact ise gene cluster. We also examined coexpression using the fdx gene in the ise gene cluster as a reporter Fd and identified iscS, hscB, hscA, and ORF3 as being involved in the assembly of the [2Fe-2S] cluster in this protein. We propose a model in which the fdx gene product functions as an intermediate site for Fe-S cluster assembly.