Transcriptional and post-transcriptional regulation by nickel of sodN gene encoding nickel-containing superoxide dismutase from Streptomyces coelicolor Muller
Transcriptional and post-transcriptional regulation by nickel of sodN gene encoding nickel-containing superoxide dismutase from Streptomyces coelicolor Muller
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DOI：
10.1046/j.1365-2958.1998.00674.x
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发表时间：
1998-01-01
影响因子：
3.6
通讯作者：
Roe, JH
中科院分区：
文献类型：
--
作者：
Kim, EJ;Chung, HJ;Roe, JH
在几种链霉菌属中发现了一种新型的含有镍作为辅因子的超氧化物歧化酶 (NiSOD)。NiSOD (sodN) 的基因是使用根据纯化酶的 N 端肽序列设计的简并寡核苷酸探针从天蓝链霉菌中克隆的。它编码 131 个氨基酸 (14703 Da) 的多肽，与其他已知蛋白质没有任何明显的序列相似性。纯化的 NiSOD 的 N 末端位于推导的开放阅读框 (ORF) 起始密码子下游 14 个氨基酸处，表明参与了蛋白质加工。加工后的多肽的分子量预计为 13201 Da，与纯化的 NiSOD (13.4 kDa) 非常一致。通过S1作图和引物延伸分析确定sodN基因的转录起始位点。 Ni2+ 调节 NiSOD 多肽的合成。 sodN mRNA 5' 和 3' 末端的 S1 作图显示，Ni2+ 使单顺反子 sodN mRNA 的水平增加了九倍以上，而没有改变其半衰期，从而证明 Ni2+ 调节转录。在缺乏足够 Ni2+ 的情况下，变青链球菌中克隆的 sodN 基因可产生具有很小 SOD 活性的前体和加工过的 NiSOD 多肽；然而，添加Ni2+后，形成仅由加工过的多肽组成的活性NiSOD。全长 sodN 基因在大肠杆菌中的表达产生了 NiSOD 多肽，即使在 Ni2+ 存在下也没有任何 Son 活性。然而，从 sodN 基因中删除编码 N 端 14 个氨基酸的核苷酸允许在大肠杆菌中产生活性 NiSOD，这表明需要 N 端加工来产生活性 NiSOD。这些结果揭示了镍作为天蓝色链球菌中控制 sodN 转录和蛋白质加工的多方面调节剂的独特作用，以及作为催化辅助因子的作用。
A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in several Streptomyces spp, The gene for NiSOD (sodN) was cloned from S. coelicolor Muller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins. The N-terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa). The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis. Ni2+ regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5' and 3' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription. Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the cloned sodN gene in S. lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full-length sodN gene in E. coli produced NiSOD polypeptide without any Son activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active NiSOD in E. coli, indicating that N-terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator in S. coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.