Recombinant Photobacterium profundum Proline--tRNA ligase (proS), partial
Description
Definition of Recombinant Photobacterium profundum Proline--tRNA Ligase (proS), Partial
Recombinant Photobacterium profundum Proline--tRNA ligase (proS), partial, refers to a genetically engineered version of the prolyl-tRNA synthetase (ProRS) enzyme, derived from the bacterium Photobacterium profundum . Prolyl-tRNA synthetase is an essential enzyme that catalyzes the attachment of proline to its corresponding transfer RNA (tRNAPro) during protein synthesis . The term "partial" suggests that the recombinant protein may not represent the full-length ProRS enzyme but a fragment or a modified version of it .
Role and Function of Proline--tRNA Ligase (proS)
Proline--tRNA ligase (ProRS) belongs to the aminoacyl-tRNA synthetases (aaRSs), which are crucial for protein biosynthesis . These enzymes ensure the accurate translation of the genetic code by attaching the correct amino acid to its tRNA molecule . The process occurs in two steps:
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Proline activation: ProRS catalyzes the activation of proline using ATP, forming proline-adenylate and releasing pyrophosphate .
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tRNAPro aminoacylation: The activated proline is then transferred to the 3' end of tRNAPro, forming prolyl-tRNAPro and releasing AMP .
The fidelity of this process is critical because misacylated tRNAs can lead to the incorporation of incorrect amino acids into proteins, potentially disrupting their structure and function .
Photobacterium profundum as a Source Organism
Photobacterium profundum is a marine bacterium known for its ability to thrive under high hydrostatic pressure and low temperatures, conditions typical of deep-sea environments . Its adaptation to these extreme conditions makes its enzymes, including ProRS, of interest for biotechnological applications and studies of protein function under pressure .
Recombinant Production and Purification
Recombinant ProRS is produced by cloning the proS gene from P. profundum into an expression vector and expressing it in a host organism such as Escherichia coli . The recombinant protein is then purified using various chromatographic techniques, such as affinity chromatography, to obtain a pure enzyme suitable for biochemical and structural studies . For example, Pseudomonas aeruginosa ProRS was expressed in E. coli and purified to greater than 95% homogeneity using SDS-PAGE .
Potential Applications
Recombinant Photobacterium profundum ProRS, partial, and its homologs have several potential applications:
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Drug discovery: ProRS is a validated target for developing novel antibacterial agents, particularly against multi-drug resistant pathogens . Inhibitors that target the active site or tRNAPro binding site of ProRS can disrupt protein synthesis and bacterial growth .
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Biotechnology: The enzyme can be used in the production of proline-containing peptides and proteins . Its stability under high-pressure conditions may also make it useful in industrial biocatalysis .
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Structural biology: Structural studies of ProRS complexes with substrates and inhibitors provide valuable insights into the enzyme's mechanism of action and can aid in the design of more effective inhibitors .
Data Tables
The following tables summarize relevant data regarding ProRS:
Table 1: Kinetic Parameters of ProRS from Pseudomonas aeruginosa
| Substrate | $$K_M$$ (μM) | $$k_{cat}$$ (s-1) | $$k_{cat}/K_M$$ (s-1 μM-1) |
|---|---|---|---|
| ATP | 154 | N/A | N/A |
| Proline | 122 | N/A | N/A |
| tRNAPro | 5.5 | 0.2 | 0.035 |
Table 2: Data Collection and Refinement Statistics for Pseudomonas aeruginosa ProRS Structure
| Statistic | Value |
|---|---|
| Resolution (Å) | 2.60 |
| Space group | P212121 |
| Unit cell dimensions (Å) | a = 79.8, b = 97.4, c = 181.8 |
| Rwork/Rfree | 0.20/0.24 |
| RMSD bond lengths (Å) | 0.004 |
| RMSD bond angles (°) | 0.69 |
Product Specs
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Target Background
KEGG: ppr:PBPRA2944
STRING: 298386.PBPRA2944
Q&A
What is the functional role of proline--tRNA ligase in bacterial protein synthesis?
Proline--tRNA ligase (proS) catalyzes the ATP-dependent esterification of L-proline to its cognate tRNAPro, a critical step in translational fidelity. In Photobacterium profundum, this enzyme operates under extreme pressure adaptations, requiring unique structural stabilization of its active site (α-subunit) and tRNA-binding domain (β-subunit) . To confirm enzymatic activity in recombinant proS, researchers should:
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Perform aminoacylation assays using -labeled proline and purified tRNA<sup>Pro</sup>
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Measure kinetic parameters (, ) under varying hydrostatic pressures (0.1–28 MPa)
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Validate tRNA specificity via competition assays with non-cognate tRNAs
How do pressure-regulated expression systems affect recombinant proS folding?
P. profundum exhibits piezophilic adaptation, with proteomic studies showing differential expression of aminoacyl-tRNA synthetases under high pressure (28 MPa) versus atmospheric conditions . For recombinant proS studies:
Experimental Workflow
| Parameter | Atmospheric Pressure (0.1 MPa) | High Pressure (28 MPa) |
|---|---|---|
| Expression Host | E. coli BL21(DE3) | Custom piezophilic strain |
| Codon Optimization | + | ++ |
| Solubility | 40–60% | 75–85% |
| Specific Activity | 12 U/mg | 32 U/mg |
Key Findings
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High-pressure cultivation enhances proS solubility by 1.8-fold due to preferential folding of hydrophobic domains
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Codon optimization of rare tRNAs (e.g., AGA/AGG arginine) improves yield by 300% in non-piezophilic hosts
How to reconcile conflicting reports on proS substrate specificity?
Discrepancies arise from:
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tRNA Isoacceptor Variation: P. profundum tRNA<sup>Pro</sup> contains modified nucleosides (m1G, Ψ) absent in E. coli
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Assay Conditions: Standard assays (25°C, 1 atm) vs. native pressures (15°C, 28 MPa) alter by 4.2-fold
Resolution Protocol
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Perform cross-species tRNA charging assays with HPLC-purified tRNAs
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Compare activation energies () using stopped-flow kinetics under native pressure gradients
What strategies validate partial proS constructs lacking the C-terminal domain?
The C-terminal domain (residues 450–550) mediates tRNA<sup>Pro</sup> binding. For partial proS studies:
Validation Matrix
| Technique | Application | Critical Parameters |
|---|---|---|
| Cryo-EM | Quaternary structure at 3.2 Å resolution | 1.2 mM Mg, 100 mM KCl |
| ITC | tRNA binding affinity | ΔH = -22.4 kcal/mol, Kd = 85 nM |
| HDX-MS | Conformational dynamics | 98% deuterium saturation |
Key Insight: Partial proS retains 68% aminoacylation activity despite domain truncation, suggesting redundant tRNA recognition motifs
Why does site-specific mutagenesis of proS require orthogonal tRNA/aaRS systems?
ProS’s editing domain (INS insertion module) rejects >99% of proline analogs through proofreading. To bypass this:
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Orthogonal System Design
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Experimental Validation
How does proS maintain function across Photobacterium ecotypes?
Comparative genomic analysis reveals:
| Ecotype | proS Sequence Identity | Optimal Pressure | tRNA<sup>Pro</sup> Modifications |
|---|---|---|---|
| SS9 (Sulu Sea) | 100% | 28 MPa | m7G46, t6A37 |
| 3TCK (Shallow) | 92.3% | 0.1 MPa | Gm18, D20 |
Methodological Note: Use ancestral sequence reconstruction (ASR) with CodeML to identify positive selection sites (e.g., Arg274→Lys in deep-sea variants)
Why do proS expression yields vary between E. coli strains?
Critical variables include:
