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 Citations (Scopus)

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

Fingerprint

malonyl-CoA decarboxylase
Carboxy-Lyases
Bombyx
Phosphorylation
Nucleopolyhedrovirus
Decarboxylation
Post Translational Protein Processing
Malonyl Coenzyme A
Acetyl Coenzyme A
Computational Biology
Catalysis
Lipid Metabolism
Point Mutation

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

Cite this

Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity. / Hwang, In Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin kyo; Park, Enoch Y.

In: Applied Microbiology and Biotechnology, Vol. 99, No. 21, 01.11.2015, p. 8977-8986.

Research output: Contribution to journalArticle

Hwang, In Wook ; Makishima, Yu ; Suzuki, Tomohiro ; Kato, Tatsuya ; Park, Sungjo ; Terzic, Andre ; Chung, Shin kyo ; Park, Enoch Y. / Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity. In: Applied Microbiology and Biotechnology. 2015 ; Vol. 99, No. 21. pp. 8977-8986.
@article{6c2f0e473cf64be08073e20e8609efe2,
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.",
keywords = "Bombyx mori nucleopolyhedrovirus, Human malonyl-CoA decarboxylase (hMCD), Lipid metabolism, Phosphorylation/dephosphorylation, Silkworm, Site-directed mutagenesis",
author = "Hwang, {In Wook} and Yu Makishima and Tomohiro Suzuki and Tatsuya Kato and Sungjo Park and Andre Terzic and Chung, {Shin kyo} and Park, {Enoch Y.}",
year = "2015",
month = "11",
day = "1",
doi = "10.1007/s00253-015-6687-x",
language = "English (US)",
volume = "99",
pages = "8977--8986",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer Verlag",
number = "21",

}

TY - JOUR

T1 - Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity

AU - Hwang, In Wook

AU - Makishima, Yu

AU - Suzuki, Tomohiro

AU - Kato, Tatsuya

AU - Park, Sungjo

AU - Terzic, Andre

AU - Chung, Shin kyo

AU - Park, Enoch Y.

PY - 2015/11/1

Y1 - 2015/11/1

N2 - 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.

AB - 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.

KW - Bombyx mori nucleopolyhedrovirus

KW - Human malonyl-CoA decarboxylase (hMCD)

KW - Lipid metabolism

KW - Phosphorylation/dephosphorylation

KW - Silkworm

KW - Site-directed mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=84945482859&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84945482859&partnerID=8YFLogxK

U2 - 10.1007/s00253-015-6687-x

DO - 10.1007/s00253-015-6687-x

M3 - Article

C2 - 26004805

AN - SCOPUS:84945482859

VL - 99

SP - 8977

EP - 8986

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 21

ER -