Biotechnologically relevant enzymes and proteins

Applied Microbiology and Biotechnology

, Volume 98, Issue 19, pp 8201-8209

Open Access This content is freely available online to anyone, anywhere at any time.

Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation

  • In-Wook HwangAffiliated withLaboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University
  • , Yu MakishimaAffiliated withLaboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University
  • , Tatsuya KatoAffiliated withLaboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka UniversityLaboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University
  • , Sungjo ParkAffiliated withCenter for Regenerative Medicine, Mayo ClinicMarriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, and Medical Genetics, Mayo Clinic
  • , Andre TerzicAffiliated withCenter for Regenerative Medicine, Mayo ClinicMarriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, and Medical Genetics, Mayo Clinic Email author 
  • , Enoch Y. ParkAffiliated withLaboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka UniversityLaboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka UniversityLaboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University Email author 

Abstract

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

Keywords

Human acetyl-CoA carboxylase 2 (hACC2) Phosphorylation/dephosphorylation Lipid metabolism Silkworm Bombyx mori nucleopolyhedrovirus