Recombinant Xenopus laevis Acyl-CoA synthetase family member 3, mitochondrial (acsf3), partial

What is the functional role of ACSF3 in mitochondrial metabolism?

ACSF3 catalyzes ATP-dependent ligation of CoA to malonate/methylmalonate, producing malonyl-CoA/methylmalonyl-CoA for mitochondrial fatty acid synthesis and protein malonylation . Key experimental approaches:

• Metabolic flux assays: Use 13C-labeled malonate to trace incorporation into acetyl-CoA and TCA cycle intermediates via LC-MS/MS .

• CRISPR/Cas9 KO models: Assess metabolic disruptions (e.g., reduced acetyl-L-carnitine m+1 isotopomers) and lysine malonylation defects .

How does recombinant Xenopus laevis ACSF3 differ from mammalian orthologs?

X. laevis ACSF3 shares conserved malonyl-CoA synthetase activity but exhibits species-specific regulatory features:

Methodological note: Perform cross-species activity assays using pH-optimized buffers (pH 6.5–9.5) .

What are common pitfalls in recombinant ACSF3 expression?

• Host system limitations: E. coli lacks mitochondrial chaperones, risking improper folding; mammalian systems yield <85% purity without affinity tags .

How to resolve discrepancies in ACSF3-dependent metabolic flux data?

Contradictions arise from model-specific malonate availability or compensatory pathways:

• In vitro vs. in vivo models: X. laevis oocytes show higher malonate detoxification capacity than human cell lines .

• Data reconciliation strategy:

  • Quantify intracellular malonate via GC-MS .

  • Inhibit malonyl-CoA decarboxylase (MLYCD) to isolate ACSF3-specific effects .

What methods validate ACSF3's role in post-translational modifications?

• Malonylation profiling:

  • Immunoprecipitate mitochondrial lysates with anti-malonyllysine antibodies .

  • Identify targets via LC-MS/MS (e.g., SDHA, ATP5A1) .

• Functional validation: CRISPR rescue experiments with catalytically dead mutants (e.g., ACSF3-E337Q) .

How to design experiments linking ACSF3 to disease models?

• Methylmalonic acidemia (MMA): Use X. laevis embryos injected with ACSF3 siRNA :

  Parameter	Control	siRNA-Treated
  Malonyl-CoA (nmol/g)	12.4 ± 1.2	3.1 ± 0.8*
  Survival rate (%)	98	62
  *p < 0.01, n = 50 embryos/group .

• Endpoint selection: Prioritize TCA cycle intermediates (citrate, α-KG) and acyl-carnitines .

What computational tools predict ACSF3 interaction networks?

• Pathway mapping: Use KEGG (map01212) and Reactome (R-HSA-2046104) .

• Structural modeling: Alphafold2-predicted ACSF3 structure (AF-Q4G176) identifies ATP-binding pockets (residues 210-230) .

Methodological Best Practices

• Purification: Opt for mammalian expression systems with FLAG-tags for >95% purity (Protein G affinity) .

• Activity normalization: Correct for mitochondrial extraction efficiency using citrate synthase activity as a control .