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Characterization of recombinant glutathione reductase from Antarctic yeast Rhodotorula mucilaginosa

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Abstract

Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide, which helps to maintain a cellular reducing environment during stress in organisms. However, GR of polar yeast has not been well-characterized so far. To fully understand the molecular and enzymatic properties of GR from extreme area and broaden its knowledge, a cDNA-encoding GR from Antarctic sea-ice yeast Rhodotorula mucilaginosa (designated as RmGR) was cloned and expressed in Escherichia coli. The open reading frame of RmGR was 1500 bp, encoding an enzyme of 499 amino acids with a predicted pI of 6.07 and molecular weight of 54.8 kDa. SDS-PAGE and gel filtration analysis results showed that the RmGR was a homodimer. Conserved sequence analysis revealed that RmGR behaved typical characteristics of GR, containing a pyridine nucleotide-disulfide oxidoreductase active site, a flavin adenine dinucleotide (FAD), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs and two glutathione oxidized binding motifs. The recombinant enzyme displayed relatively high stability at pH 3.0–6.0 and 20–40 °C with optimal enzymatic activity at 30 °C and pH 7.5. Real-time quantitative PCR data showed that the expression of RmGR gene was upregulated after copper treatment of yeast cells. Moreover, RmGR-transformed E. coli exhibited stronger growth profiles than cells with an empty vector after copper and cadmium treatment. Our results demonstrated the possible function of RmGR in adaptation to heavy metals and its potential application in heterologous expression systems.

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Acknowledgements

This work was supported by Key Technologies R & D Program of Shandong (Grant Nos. 2016ZDJQ0206, 2019GHY112047), Science and Technology Project of Weihai (WH20140209), and Natural Scientific Research Innovation Foundation in Harbin Institute of Technology (Grant No. HIT.IBRSEM.2013037 and HIT.NSRIF.2016085).

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Authors and Affiliations

  1. School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, Shandong, People’s Republic of China

    Xiaofei Wang, Cuijuan Shi, Guofu Chen, Jie Jiang, Yun Ju, Ruiqi Wang, Guangfeng Kan, Haoruo Wei & FanFan Zhu

  2. Key Laboratory of Tobacco Pest Monitoring & Integrated Management, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, Shandong, People’s Republic of China

    Chengsheng Zhang

  3. Wendeng Osteopath Hospital, Wendeng, Shandong, People’s Republic of China

    Yongping Qiao

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  1. Xiaofei Wang
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Correspondence to Guangfeng Kan.

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Wang, X., Shi, C., Chen, G. et al. Characterization of recombinant glutathione reductase from Antarctic yeast Rhodotorula mucilaginosa. Polar Biol 42, 2249–2258 (2019). https://doi.org/10.1007/s00300-019-02603-3

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  • DOI: https://doi.org/10.1007/s00300-019-02603-3

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