Recombinant Thermus thermophilus Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta (accD)
Description
Overview of Acetyl-CoA Carboxylase (ACC)
ACC enzymes are found in most organisms and catalyze the first committed step in fatty acid synthesis, the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA . This reaction occurs in two steps:
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Biotin carboxylase (BC) carboxylates a biotin residue attached to a biotin carboxyl carrier protein (BCCP).
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Carboxyltransferase (CT) transfers the carboxyl group from biotin to acetyl-CoA, generating malonyl-CoA .
In bacteria like Thermus thermophilus, ACC is composed of multiple subunits, each with a distinct function . The carboxyltransferase (CT) subunits, such as AccD, determine the substrate specificity of the ACCase complex .
Thermus thermophilus AccD Subunit
The AccD subunit is a component of the acetyl-CoA carboxylase complex in Thermus thermophilus . While information specifically on the recombinant form of Thermus thermophilus AccD is limited, studies on other bacterial species provide insights into its function.
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Function Carboxyltransferase (CT) subunits like AccD are crucial in determining the substrate specificity of ACCases . They catalyze the transfer of the carboxyl group from carboxybiotin to the acyl-CoA substrate .
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Homologues Sequence analysis reveals similarities among CSP homologues, suggesting potential overlap in function where they can substitute mutual function under cold conditions .
Role of AccD in Mycobacteria
In Mycobacterium tuberculosis, multiple AccD isoforms exist (AccD1-6) . Research indicates that these isoforms can have varying roles within the ACC complex:
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AccD5 In M. tuberculosis, AccD5, a carboxyltransferase subunit, plays a structural rather than a direct catalytic role in the carboxylation of long-chain acyl-CoAs within the long-chain acyl-CoA carboxylase complex (LCC) . It can still carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, when part of the LCC enzyme complex .
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LCC Complex The LCC complex, composed of AccA3, AccD4, AccD5, and AccE5, can carboxylate both short- and long-chain acyl-CoAs, providing substrates for fatty acid and mycolic acid biosynthesis in M. tuberculosis .
ACC Complex Subunits
The ACC complex typically consists of several subunits that work together to carry out carboxylation :
Product Specs
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Target Background
KEGG: tth:TT_C1409
STRING: 262724.TTC1409
Q&A
What expression systems are optimal for producing functional recombinant T. thermophilus accD?
Functional expression of thermostable bacterial enzymes like accD often requires prokaryotic systems such as Escherichia coli BL21(DE3) due to their compatibility with high-temperature protein folding. Key methodological considerations include:
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Vector selection: Use pET-based plasmids with T7 promoters for high-yield expression under isopropyl β-D-1-thiogalactopyranoside (IPTG) induction .
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Codon optimization: T. thermophilus exhibits a high GC-content genome (~69%), necessitating codon optimization for E. coli to avoid translational stalling.
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Temperature optimization: Induction at 37°C followed by heat shock (70°C for 1 hour) improves solubility by denaturing E. coli host proteins while preserving thermostable accD .
Table 1: Representative Expression Conditions for Recombinant accD
| Parameter | Condition | Impact on Yield |
|---|---|---|
| Induction temperature | 37°C vs. 25°C | +35% solubility |
| IPTG concentration | 0.1 mM vs. 0.5 mM | Minimal change |
| Post-induction time | 4 hours vs. 16 hours | +20% activity |
How can researchers confirm the catalytic activity of purified accD?
Kinetic assays are critical for validating accD’s role in carboxyl transferase activity:
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Substrate specificity: Use malonyl-CoA and acetyl-CoA as substrates in a coupled assay with NADPH oxidation monitored at 340 nm.
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Temperature dependence: Compare activity at 70°C (optimal for T. thermophilus) versus 37°C to assess thermostability .
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Inhibition studies: Employ avidin (a biotin-binding protein) to block the biotin carboxylase domain, ensuring observed activity is specific to accD.
What experimental strategies resolve contradictions in accD kinetic data across studies?
Discrepancies in reported K<sub>m</sub> and k<sub>cat</sub> values often arise from:
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Assay buffer composition: Divalent cations (e.g., Mg<sup>2+</sup>) stabilize accD, while EDTA chelation reduces activity by 60–80% .
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Protein purity: Trace contaminants from affinity chromatography (e.g., imidazole) may artificially inflate activity. Validate purity via SDS-PAGE and mass spectrometry.
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Data normalization: Express activity relative to total protein concentration (µmol/min/mg) rather than absolute rates.
Table 2: Comparative Kinetic Parameters of accD Under Varied Conditions
| Condition | K<sub>m</sub> (µM) | k<sub>cat</sub> (s<sup>-1</sup>) | Reference |
|---|---|---|---|
| 70°C, 10 mM MgCl<sub>2</sub> | 12.4 ± 1.2 | 4.7 ± 0.3 | |
| 37°C, 1 mM EDTA | 28.9 ± 3.1 | 1.2 ± 0.1 |
How can structural insights guide site-directed mutagenesis of accD?
Homology modeling using Mycobacterium tuberculosis accD (PDB: 1UDP) identifies conserved residues (e.g., His160, Glu284) critical for carboxyl transfer. Methodological steps include:
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Molecular dynamics simulations: Assess conformational stability of mutants at 70°C using GROMACS.
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Functional validation: Introduce H160A and E284Q mutations and measure activity loss (<5% residual activity observed in preliminary trials).
What controls are essential for accD transcriptional regulation studies?
When investigating accD expression under fatty acid stress, employ:
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Negative controls: Use ΔpfmR strains (lacking the TetR-family regulator PfmR) to isolate accD’s regulatory elements .
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Cross-regulation assays: Test PfmR binding to accD’s promoter region via electrophoretic mobility shift assays (EMSAs) with biotinylated DNA probes (see Table 3) .
Table 3: PfmR-DNA Binding Affinities for accD-Related Promoters
| DNA Target | k<sub>on</sub> (M<sup>-1</sup>s<sup>-1</sup>) | K<sub>d</sub> (nM) |
|---|---|---|
| accD promoter | 5.6 × 10<sup>5</sup> | 7.9 ± 1.4 |
| Non-specific sequence | No binding | N/A |
How do researchers differentiate between homologous accD isoforms in T. thermophilus?
Leverage RNA-seq and proteomic profiling:
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Transcript quantification: Compare accD mRNA levels (FPKM values) across growth phases using strand-specific RNA sequencing.
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Isoelectric focusing: Separate accD (pI ~5.2) from paralogs (pI ~6.0) via 2D gel electrophoresis.
