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Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity

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Abstract

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (−200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation.

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Acknowledgments

This work was supported partly by the Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation from Japan Society for the Promotion of Science (JSPS), Japan, National Institute of Health, and Marriott Heart Disease Research Program at the Mayo Clinic. A.T. holds the Marriott Family Professorship in Cardiovascular Diseases Research.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

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

    In-Wook Hwang & Enoch Y. Park

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

    Yu Makishima, Tatsuya Kato & Enoch Y. Park

  3. Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan

    Tomohiro Suzuki

  4. 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

  5. Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA

    Sungjo Park & Andre Terzic

  6. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, and Medical Genetics, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA

    Sungjo Park & Andre Terzic

  7. School of Food Science and Biotechnology, Kyungpook National University, Daegu, 702-701, Republic of Korea

    Shin-kyo Chung

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  1. In-Wook Hwang
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  2. Yu Makishima
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Correspondence to Enoch Y. Park.

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Hwang, IW., Makishima, Y., Suzuki, T. et al. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity. Appl Microbiol Biotechnol 99, 8977–8986 (2015). https://doi.org/10.1007/s00253-015-6687-x

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