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.

Conventional and unconventional secretory proteins expressed with silkworm bombyxin signal peptide display functional fidelity

“…Bacmid injected larvae were reared at room temperature for 6–7 days, and then the fat body was collected by dissection, and immediately frozen at −70 °C until further analysis. Protein purification was carried out at 4 °C to minimize aggregation and protease activity 38 – 40 . Larval fat bodies were homogenized in 10 mL of lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7.5 and 0.1% Triton X-100) containing an EDTA-free protease inhibitor tablet (Roche) using a Dounce tissue grinder.…”

Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

“…The recombinant BmNPV bacmid DNA is directly injected into silkworm larvae and pupae for foreign gene expression 25 , 26 . Using this system, we have successfully produced cellular, mitochondrial, and membrane proteins exhibiting proper folding and post-translational modifications 27 – 30 .…”

Identification of the active site of human mitochondrial malonyl‐coenzyme a decarboxylase: A combined computational study

“…These MCDs derived from different species have conserved amino acid sequences, but they do not share recognizable homology with other protein in the sequence database. Decrease or deficiency of MCD activity induces the accumulation of long‐chain acetyl‐CoA, which may activate peroxisome proliferator‐activated receptor (PPAR) nuclear receptors . In humans, deficiency of malonyl‐CoA decarboxylase causes a rare autosomal recessive metabolic disorder, which has been characterized by a variable phenotype of developmental delay, cardiomyopathy, hypoglycemia, metabolic acidosis, and malonic aciduria …”

“…Decrease or deficiency of MCD activity induces the accumulation of long-chain acetyl-CoA, which may activate peroxisome proliferator-activated receptor (PPAR) nuclear receptors. 5,12 In humans, deficiency of malonyl-CoA decarboxylase causes a rare autosomal recessive metabolic disorder, which has been characterized by a variable phenotype of developmental delay, cardiomyopathy, hypoglycemia, metabolic acidosis, and malonic aciduria. [13][14][15] In recent years, the reports about purification of several animal and bacterial MCDs have been released, [16][17][18] and the crystal structure of a bacterial MCD was also resolved by Jung et al 19 The available crystal structures from human mitochondrial and peroxisomal MCDs have been reported by Froese et al 20 and Aparicio et al 21 in 2013, respectively.…”