Transcriptional and post‐transcriptional regulation by nickel ofsodNgene encoding nickel‐containing superoxide dismutase fromStreptomyces coelicolorMüller

Open Access
Publisher Website
Google Scholar
Abstract
A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in severalStreptomycesspp. The gene for NiSOD (sodN ) was cloned fromS.coelicolorMüller 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 thesodNgene was determined by S1 mapping and primer extension analysis. Ni2+regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5′ and 3′ ends ofsodNmRNA revealed that Ni2+increased the level of monocistronicsodNmRNA 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 clonedsodNgene inS.lividansin the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full‐lengthsodNgene inE.coliproduced NiSOD polypeptide without any SOD activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N‐terminal 14 amino acids from thesodNgene allowed the production of active NiSOD inE.coli, indicating that N‐terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator inS.coelicolorcontrollingsodNtranscription and protein processing, as well as acting as a catalytic cofactor.