title: Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.
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.
authors.affiliation: Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. gunryung21@hanmail.net.
authors.forename: In-Wook
authors.initials: IW
authors.lastname: Hwang
authors.affiliation: Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. mmm.yu@live.jp.
authors.forename: Yu
authors.initials: Y
authors.lastname: Makishima
authors.affiliation: Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. atsuzuk@ipc.shizuoka.ac.jp.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. atkato@ipc.shizuoka.ac.jp.|Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. atkato@ipc.shizuoka.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kato
authors.affiliation: Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA. park.sungjo@mayo.edu.|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. park.sungjo@mayo.edu.
authors.forename: Sungjo
authors.initials: S
authors.lastname: Park
authors.affiliation: Center for Regenerative Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA. terzic.andre@mayo.edu.|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. terzic.andre@mayo.edu.
authors.forename: Andre
authors.initials: A
authors.lastname: Terzic
authors.affiliation: School of Food Science and Biotechnology, Kyungpook National University, Daegu, 702-701, Republic of Korea. kchung@knu.ac.kr.
authors.forename: Shin-Kyo
authors.initials: SK
authors.lastname: Chung
authors.affiliation: Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. park.enoch@shizuoka.ac.jp.|Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. park.enoch@shizuoka.ac.jp.|Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. park.enoch@shizuoka.ac.jp.
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001711:Biotransformation; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase
keywords: Bombyx mori nucleopolyhedrovirus; Human malonyl-CoA decarboxylase (hMCD); Lipid metabolism; Phosphorylation/dephosphorylation; Silkworm; Site-directed mutagenesis