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Molecular cloning, purification, and characterization of a superoxide dismutase from a fast-growing Mycobacterium sp. Strain JC1 DSM 3803

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Abstract

A cytosolic superoxide dismutase (SOD) was purified and characterized from a fast-growing Mycobacterium sp. strain JC1 DSM 3803 grown on methanol. The native molecular weight of the purified SOD was estimated to be 48 kDa. SDS-PAGE revealed a subunit of 23 kDa, indicating that the enzyme is a homodimer. The enzyme activity was inhibited by H2O2 and azide. The purified SOD contained 1.12 and 0.56 g-atom of Mn and Fe per mol of enzyme, respectively, suggesting that it may be a Fe/Mn cambialistic SOD. The apo-SOD reconstitution study revealed that Mn salts were more specific than Fe salts in the SOD activity. The gene encoding the SOD was identified from the JC1 cosmid genomic library by PCR screening protocol. The cloned gene, sodA, had an open reading frame (ORF) of 624 nt, encoding a protein with a calculated molecular weight of 22,930 Da and pi of 5.33. The deduced SodA sequence exhibited 97.6% identity with that of Mycobacterium fortuitum Mn-SOD and clustered with other mycobacterial Mn-SODs. A webtool analysis on the basis of SOD sequence and structure homologies predicted the SOD as a tetrameric Mn-SOD, suggesting that the protein is a dimeric Mn-SOD having tetramer-specific sequence and structure characteristics.

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Author notes

  1. Hyun-Il Lee

    Present address: Department of Orthopedic Surgery, Samsung Medical Center, Seoul, 135-710, Republic of Korea

Authors and Affiliations

  1. Department of Biochemistry, Graduate School of Medicine, Konkuk University, Chungju, 380-701, Republic of Korea

    Ji-Sun Nam, Jee-Hyun Yoon, Hyun-Il Lee & Young-Tae Ro

  2. Department of Environmental Engineering, Chosun University, Gwangju, 501-759, Republic of Korea

    Si Wouk Kim

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  1. Ji-Sun Nam
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  2. Jee-Hyun Yoon
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  5. Young-Tae Ro
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Correspondence to Young-Tae Ro.

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Nam, JS., Yoon, JH., Lee, HI. et al. Molecular cloning, purification, and characterization of a superoxide dismutase from a fast-growing Mycobacterium sp. Strain JC1 DSM 3803. J Microbiol. 49, 399–406 (2011). https://doi.org/10.1007/s12275-011-1046-9

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