Molecular Cloning, Purification, and Characterization of a Superoxide Dismutase from a Fast-Growing Mycobacterium sp. Strain JC1 DSM 3803

Ji-Sun Nam; Jee-Hyun Yoon; Hyun-Il Lee; Si Wouk Kim; Young-Tae Ro

doi:10.1007/s12275-011-1046-9

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Research Support, Non-U.S. Gov't
Molecular Cloning, Purification, and Characterization of a Superoxide Dismutase from a Fast-Growing Mycobacterium sp. Strain JC1 DSM 3803: Ji-Sun Nam ¹, Jee-Hyun Yoon ¹, Hyun-Il Lee ¹, Si Wouk Kim ², Young-Tae Ro ¹; Journal of Microbiology 2011;49(3):399-406.
DOI: https://doi.org/10.1007/s12275-011-1046-9
Published online: June 30, 2011

¹Department of Biochemistry, Graduate School of Medicine, Konkuk University, Chungju 380-701, Republic of Korea, ²Department of Environmental Engineering, Chosun University, Gwangju 501-759, Republic of Korea

Corresponding author: Young-Tae Ro , Tel: +82-2-2030-7825,

Received: 28 January 2011 • Accepted: 16 February 2011

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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.

Keywords: Mn-SOD;Mycobacterium;PCR gene screening;sequence analysis;superoxide dismutase

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Molecular Cloning, Purification, and Characterization of a Superoxide Dismutase from a Fast-Growing Mycobacterium sp. Strain JC1 DSM 3803

J. Microbiol. 2011;49(3):399-406. Published online June 30, 2011

DOI: https://doi.org/10.1007/s12275-011-1046-9

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