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Functional Analysis of Ribonucleotide Reductase from Cordyceps militaris Expressed in Escherichia coli

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Abstract

Cordyceps militaris produces cordycepin (3′-deoxyadenosine), which has various activities, including anti-oxidant, anti-tumoral, anti-viral, and anti-inflammatory. Ribonucleotide reductase (RNR) seems to be a candidate to produce cordycepin in C. militaris because RNR catalyzes the reduction of nucleotides to 2′-deoxynucleotides, whose structures are similar to that of cordycepin. However, the role of RNR has not been confirmed yet. In this study, complementary DNAs (cDNAs) of C. militaris RNR (CmRNR) large and small subunits (CmR1 and CmR2) were cloned from C. militaris NBRC9787 to investigate the function of CmRNR for its cordycepin production. C. militaris NBRC9787 began to produce cordycepin when grown in a liquid surface culture in medium composed of glucose and yeast extract for 15 days. CmR1 cDNA and CmR2 cDNA were obtained from its genomic DNA and from total RNA extracted from its mycelia after cultivation for 21 days, respectively. Recombinant CmR1 and CmR2 were expressed individually in Escherichia coli and purified. Purified recombinant CmR1 and CmR2 showed RNR activity toward adenosine diphosphate (ADP) only when two subunits were mixed but only show the reduction of ADP to 2′-deoxyADP. These results indicate that the pathway from ADP to 3′deoxyADP via CmRNR does not exist in C. militaris and cordycepin production in C. militaris may be mediated by other enzymes.

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Acknowledgements

Authors thank Mr. Yasuhiro Kojima who performed the gene manipulation and HPLC measurement.

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Authors and Affiliations

  1. Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Tatsuya Kato, Suparmin Ahmad & Enoch Y. Park

  2. Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan

    Tatsuya Kato & Enoch Y. Park

  3. Laboratory of Biotechnology, Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Tatsuya Kato & Enoch Y. Park

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  1. Tatsuya Kato
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  2. Suparmin Ahmad
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Correspondence to Enoch Y. Park.

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Kato, T., Ahmad, S. & Park, E.Y. Functional Analysis of Ribonucleotide Reductase from Cordyceps militaris Expressed in Escherichia coli . Appl Biochem Biotechnol 182, 1307–1317 (2017). https://doi.org/10.1007/s12010-017-2400-0

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