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

In Wook Hwang, Yu Makishima, Tomohiro Suzuki, Tatsuya Kato, Sungjo Park, Andre Terzic, Shin kyo Chung, Enoch Y. Park

Research output: Contribution to journalArticle

2 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)8977-8986
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume99
Issue number21
DOIs
StatePublished - Nov 1 2015

Keywords

  • Bombyx mori nucleopolyhedrovirus
  • Human malonyl-CoA decarboxylase (hMCD)
  • Lipid metabolism
  • Phosphorylation/dephosphorylation
  • Silkworm
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

Fingerprint Dive into the research topics of 'Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity'. Together they form a unique fingerprint.

Cite this