caa a22 4500 605505462 CHVBK 20210128100622.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6687-x doi (NATIONALLICENCE)springer-10.1007/s00253-015-6687-x Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity [Elektronische Daten] [In-Wook Hwang, Yu Makishima, Tomohiro Suzuki, Tatsuya Kato, Sungjo Park, Andre Terzic, Shin-kyo Chung, Enoch Park] 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 (−200μg/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. Springer-Verlag Berlin Heidelberg, 2015 Human malonyl-CoA decarboxylase (hMCD) nationallicence Site-directed mutagenesis nationallicence Phosphorylation/dephosphorylation nationallicence Lipid metabolism nationallicence Silkworm nationallicence Bombyx mori nucleopolyhedrovirus nationallicence Hwang In-Wook Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut Makishima Yu Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut Suzuki Tomohiro Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, 422-8529, Shizuoka, Japan aut Kato Tatsuya Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut Park Sungjo Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, 55905, Rochester, MN, USA aut Terzic Andre Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, 55905, Rochester, MN, USA aut Chung Shin-kyo School of Food Science and Biotechnology, Kyungpook National University, 702-701, Daegu, Republic of Korea aut Park Enoch Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8977-8986 0175-7598 99:21<8977 2015 99 253 https://doi.org/10.1007/s00253-015-6687-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6687-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hwang In-Wook Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut NATIONALLICENCE 700 1- Makishima Yu Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut NATIONALLICENCE 700 1- Suzuki Tomohiro Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, 422-8529, Shizuoka, Japan aut NATIONALLICENCE 700 1- Kato Tatsuya Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut NATIONALLICENCE 700 1- Park Sungjo Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, 55905, Rochester, MN, USA aut NATIONALLICENCE 700 1- Terzic Andre Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, 55905, Rochester, MN, USA aut NATIONALLICENCE 700 1- Chung Shin-kyo School of Food Science and Biotechnology, Kyungpook National University, 702-701, Daegu, Republic of Korea aut NATIONALLICENCE 700 1- Park Enoch Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, 422-8529, Shizuoka, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8977-8986 0175-7598 99:21<8977 2015 99 253