Recombinant 3-ketoacyl-CoA thiolase (fadI), partial
Description
Introduction
Recombinant 3-ketoacyl-CoA thiolase (fadI), partial is a truncated form of the enzyme encoded by the fadI gene, which catalyzes the final step of fatty acid β-oxidation. This enzyme cleaves 3-ketoacyl-CoA into acetyl-CoA and a shorter fatty acyl-CoA, enabling carbon chain shortening for further metabolic processing or energy production . Recombinant versions are engineered for research and biotechnological applications, including fatty acid metabolism studies, enzyme engineering, and production of bioactive compounds .
Structure and Function
The enzyme belongs to the thiolase family (EC 2.3.1.16) and adopts a homodimeric structure with a thiolase-like fold . Its active site facilitates the thiolytic cleavage of 3-ketoacyl-CoA, a reaction critical for mitochondrial and peroxisomal fatty acid oxidation . The recombinant partial form retains catalytic activity but may lack regulatory domains, enabling focused biochemical studies .
3.1. Fatty Acid Production
Recombinant fadI is used in metabolic engineering to optimize fatty acid synthesis. For example, in Saccharomyces cerevisiae, its expression enhances the production of medium-chain fatty acids by redirecting β-oxidation intermediates .
3.2. Biofuel Development
The enzyme contributes to the biosynthesis of biofuels like n-butanol and dicarboxylic acids by enabling carbon chain elongation via Claisen condensation .
3.3. Disease Modeling
Defects in hadhb (human homolog of fadI) cause long-chain 3-ketoacyl-CoA thiolase deficiency, a rare fatty acid oxidation disorder. Recombinant fadI aids in studying this condition and validating therapeutic interventions .
4.1. Expression Systems
The enzyme is expressed in:
5.2. Engineering Advances
Structure-guided mutagenesis enhances substrate specificity and thermal stability, enabling industrial-scale biosynthesis .
5.3. Disease Implications
Mutations in hadhb disrupt mitochondrial trifunctional protein (MTP) activity, leading to cardiomyopathy and metabolic crises. Recombinant fadI models this defect for therapeutic screening .
Product Specs
Target Background
KEGG: ecv:APECO1_4224
Q&A
What is the functional role of recombinant 3-ketoacyl-CoA thiolase (fadI) in bacterial fatty acid β-oxidation?
Recombinant fadI catalyzes the thiolytic cleavage of 3-ketoacyl-CoA intermediates during β-oxidation, producing acetyl-CoA and a shortened acyl-CoA chain . Methodological verification involves:
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Gene knockout studies: Compare β-oxidation efficiency in E. coli ΔfadI mutants versus wild-type strains using radiolabeled palmitate tracers .
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Enzyme kinetics: Measure substrate specificity using acyl-CoA derivatives (C4–C16) via spectrophotometric NADH-coupled assays .
How do researchers confirm the structural integrity of recombinant fadI variants?
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Homology modeling: Align fadI sequences (e.g., E. coli fadI [GenBank NC_000913.2]) with solved thiolase structures (PDB: 1DLV) . Critical residues (Cys89, His348) are validated via:
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Site-directed mutagenesis: Replace catalytic residues and assay for loss of thiolytic activity .
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Circular dichroism: Compare α-helix/β-sheet ratios between wild-type and mutants to detect misfolding .
How should experimental designs account for parameter uncertainty in fadI kinetic studies?
Sloppy parameter models necessitate multi-pronged approaches :
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Global sensitivity analysis: Use Markov chain Monte Carlo (MCMC) sampling to identify insensitive parameters (e.g., oxygen diffusion rates in in vitro assays) .
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Optimal experimental design: Prioritize measurements that constrain predictions for acyl-CoA chain-length specificity (e.g., C8 vs. C16 substrates) .
Example contradiction resolution: Discrepancies in reported K<sub>m</sub> values for C12-CoA (15.3 μM vs. 9.8 μM ) may arise from:
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Assay temperature differences (25°C vs. 37°C)
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Cofactor concentrations (0.1 mM vs. 0.5 mM CoA-SH)
What strategies resolve conflicting data on fadI’s substrate inhibition patterns?
Substrate inhibition above 30 μM C16-CoA conflicts with linear kinetics up to 50 μM in Ralstonia eutropha homologs . Mitigation approaches:
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Competitive inhibition assays: Add decanoyl-CoA (C10) to test for active-site competition .
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Molecular dynamics simulations: Model acyl-binding tunnel flexibility under high substrate loads .
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Stop-flow kinetics: Resolve millisecond-scale conformational changes during catalysis .
How can researchers enhance fadI activity in recombinant methyl ketone biosynthesis?
Patent data outlines a three-pronged metabolic engineering strategy:
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Pathway deregulation: Delete fadR (acyl-CoA responsive repressor) to upregulate β-oxidation genes.
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Co-factor engineering: Overexpress fadD (acyl-CoA synthetase) to increase acyl-CoA pools.
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Byproduct minimization: Knock out fadB/fadJ (hydratase/dehydrogenase) to prevent β-hydroxyacyl-CoA accumulation .
What analytical techniques differentiate fadI activity from homologous thiolases (e.g., fadA)?
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Isoelectric focusing: Separate fadI (pI 5.2) and fadA (pI 5.8) using pH 4–7 gradient gels .
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Chain-length profiling: Compare activity ratios for C8-CoA:C16-CoA (fadI = 5.4:1 vs. fadA = 1.8:1) .
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Inhibitor screening: Test iodoacetamide sensitivity (fadI IC<sub>50</sub> = 12 μM vs. fadA IC<sub>50</sub> = 45 μM) .
How do researchers validate in silico predictions of fadI’s substrate channeling?
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Cysteine accessibility mapping: Use PEG-maleimide labeling under substrate-saturating conditions .
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FRET-based assays: Tag active-site residues with Cy3/Cy5 fluorophores to monitor conformational shifts .
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X-ray crystallography: Soak crystals with non-hydrolyzable acyl-CoA analogs (e.g., 3-ketooctanoyl-CoA) .
What lessons from LCKAT deficiency inform fadI functional studies?
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Cardiomyopathy models: Monitor ATP synthesis rates in fadI-deficient cardiomyocytes using <sup>13</sup>C-palmitate NMR .
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Resveratrol testing: Assess whether this polyphenol rescues β-oxidation defects in fadI<sup>-/-</sup> cell lines .
Critical finding: Ketone supplementation (D,L-3-hydroxybutyrate) improved cardiac output in LCKAT-deficient patients , suggesting analogous strategies for fadI-related metabolic disorders.
