Recombinant Photobacterium profundum tRNA 2-selenouridine synthase (selU)

Basic Research Questions

• What is tRNA 2-selenouridine synthase (SelU) in Photobacterium profundum and how does it differ from other bacterial SelU proteins?

SelU in Photobacterium profundum is an enzyme responsible for the conversion of 5-substituted 2-thiouridine (R5S2U) present in the anticodon of some bacterial tRNAs into 5-substituted 2-selenouridine (R5Se2U). The enzyme belongs to the SelU family first identified in Salmonella enterica serovar Typhimurium .

While the core function is conserved across bacterial species, P. profundum SelU likely contains adaptations for functioning under high hydrostatic pressure conditions found in deep-sea environments. P. profundum strain SS9 is a piezophile with optimal growth at 28 MPa (approximately 2,800 meters depth) . Unlike mesophilic bacteria such as E. coli, P. profundum has evolved molecular mechanisms to maintain protein function under extreme pressure conditions, which would likely extend to its SelU enzyme.

• What is the reaction mechanism of SelU and what substrates are required for activity?

SelU catalyzes a two-step process for the conversion of S2U to Se2U in RNA:

Step 1: Geranylation

• The S2U-RNA is geranylated using geranyl pyrophosphate (gePP) as the substrate

• This forms S-geranyl-2-thiouridine (geS2U-RNA) as an intermediate

Step 2: Selenation

• The geS2U-RNA intermediate is selenated using selenophosphate (SePO₃³⁻) as the selenium donor

• This yields the final 2-selenouridine (Se2U-RNA) product

Studies with E. coli SelU have demonstrated that the enzyme binds the R5S2U-tRNA substrate and catalyzes its geranylation. The resulting R5geS2U-tRNA remains bound to the enzyme and undergoes selenation in the second step. The R5Se2U-tRNA then dissociates from the enzyme . This process is likely conserved in P. profundum SelU.

• What are the structural domains of SelU and how do they contribute to its catalytic function?

SelU contains two distinct structural domains:

• N-terminal domain with rhodanese homology:

  • Contains a -Cys-X-X-Cys- active site

  • Likely involved in selenium handling during the catalytic cycle

• C-terminal P-loop domain:

  • Contains a Walker A motif for nucleotide binding

  • Includes an isoleucine-tRNA synthetase (IleS)-like helical region

  • Required for geranylation activity of the enzyme

  • Likely serves as the binding site for geranyl pyrophosphate (gePP)

These structural features work together to enable the two-step catalytic process. The P-loop domain facilitates the geranylation reaction, while the rhodanese domain is thought to be involved in selenium transfer during the selenation step.

• What factors influence SelU substrate recognition and binding affinity?

Multiple factors influence SelU substrate recognition and binding:

Microscale thermophoresis (MST) studies with E. coli SelU showed high binding affinity (micromolar range) between the enzyme and geranyl pyrophosphate, while other prenyl donors either did not bind or had affinity above 1 mM . The enzyme exhibits high substrate specificity for the prenylation reaction but shows less discrimination during the selenation step .

For P. profundum SelU, these recognition factors may be modified to accommodate high-pressure environments, potentially showing altered binding kinetics compared to mesophilic homologs.

• What expression systems are recommended for producing recombinant P. profundum SelU?

For recombinant expression of P. profundum SelU, consider the following methodological approach:

• Expression vector selection:

  • Use pMal-c5X or similar vectors for fusion with maltose-binding protein (MBP) to enhance solubility

  • Include a TEV protease cleavage site between MBP and SelU if native protein is required

• Host strain optimization:

  • E. coli BL21(DE3) for standard expression

  • E. coli Rosetta(DE3) if the gene contains rare codons

  • Growth at lower temperatures (16-20°C) after induction to enhance proper folding

• Expression conditions:

  • Induce with 0.1-0.3 mM IPTG

  • Grow at 16°C for 16-18 hours post-induction

  • Supplement media with selenium source (e.g., sodium selenite)

• Purification strategy:

  • Affinity chromatography using amylose resin for MBP-tagged protein

  • Ion exchange chromatography as a secondary purification step

  • Size-exclusion chromatography for final polishing

This expression strategy is based on successful methods used for E. coli SelU with modifications to account for potential challenges with the psychrophilic and piezophilic nature of P. profundum proteins.

Advanced Research Questions

• How does high hydrostatic pressure affect SelU activity and what methodologies can be used to study this?

High hydrostatic pressure likely affects SelU activity through multiple mechanisms:

• Structural modifications:

  • Pressure may alter protein conformation and enzyme-substrate interactions

  • The active site geometry might be optimized for function at elevated pressures in piezophilic bacteria

• Reaction kinetics:

  • According to Le Chatelier's principle, reactions with negative volume changes may be accelerated under pressure

  • The geranylation and selenation steps may have distinct pressure sensitivities

Methodological approaches for studying pressure effects:

• High-pressure enzyme assays:

  • Use a high-pressure microscopic chamber similar to those used for swimming velocity measurements of P. profundum

  • Adapt standard enzyme assays to high-pressure vessels with optical windows for spectroscopic measurements

• Comparative kinetic analysis:

  • Measure enzyme kinetics (KM and kcat) at atmospheric pressure and elevated pressures (up to 30 MPa)

  • Compare with homologous enzymes from non-piezophilic bacteria like E. coli

• Structural studies under pressure:

  • High-pressure NMR spectroscopy to detect conformational changes

  • Pressure-resolved X-ray crystallography if crystals can be obtained

P. profundum adapts to high pressure in numerous ways, including changes in gene expression and membrane composition . The SelU enzyme from this organism may possess unique adaptations for maintaining catalytic activity under deep-sea conditions.

• What analytical techniques are most effective for characterizing the kinetics of the two-step reaction catalyzed by SelU?

For comprehensive kinetic characterization of the two-step SelU reaction, multiple analytical techniques should be employed:

Step 1 (Geranylation) Analysis:

• HPLC with UV detection:

  • Monitor the disappearance of S2U-RNA and appearance of geS2U-RNA

  • Use a C18 reverse-phase column with appropriate mobile phase

• Mass spectrometry:

  • MALDI-TOF MS for intact RNA analysis

  • LC-MS/MS for detailed characterization of modified nucleosides

• Radioisotope incorporation:

  • Use [³H]-labeled geranyl pyrophosphate to track geranyl transfer

  • Quantify incorporation by scintillation counting

Step 2 (Selenation) Analysis:

• ICP-MS (Inductively Coupled Plasma Mass Spectrometry):

  • Quantify selenium incorporation into RNA

  • Provides sensitive detection of selenium content

• ¹⁹F NMR spectroscopy:

  • Incorporate a fluorine tag near the modification site

  • Monitor changes in chemical environment during selenation

Integrated Kinetic Analysis:

• Microscale thermophoresis (MST):

  • Measure binding affinities for each substrate

  • Determine the effect of pressure on binding constants

• Stopped-flow kinetics:

  • Track reaction progress in real-time

  • Resolve fast reaction steps that may be pressure-dependent

Reaction rates should be measured under varying substrate concentrations to determine Michaelis-Menten parameters (KM and kcat) for both steps of the reaction under different pressure conditions . This comprehensive approach will reveal how P. profundum SelU is adapted for function in the deep sea.

• How does SelU from P. profundum contribute to deep-sea adaptation and bacterial survival at high pressure?

The role of SelU in P. profundum's deep-sea adaptation likely involves several mechanisms:

• Enhanced translational accuracy:

  • Selenouridine modifications in the wobble position of tRNA can affect codon-anticodon interactions

  • May provide more precise translation under high-pressure conditions

  • Could compensate for pressure effects on ribosome structure and function

• Redox homeostasis:

  • Selenium-containing biomolecules often function in redox reactions

  • May help the bacterium cope with oxidative stress at high pressure

• Pressure-dependent gene regulation:

  • P. profundum utilizes different flagellar systems under high pressure

  • Similarly, selenouridine modifications might be regulated in a pressure-dependent manner

  • Could contribute to pressure-specific protein expression patterns

• Integration with other adaptation mechanisms:

  • May work synergistically with other adaptations like "piezolytes" (pressure-responsive osmolytes)

  • β-hydroxybutyrate (β-HB) and its oligomers serve as "piezolytes" in P. profundum

  • tRNA modifications could complement metabolic adaptations

This is an active research area where further investigation is needed to fully understand the relationship between tRNA modifications and piezophilic lifestyle. Studies comparing selenouridine levels in P. profundum grown at different pressures would provide valuable insights.

• What are the most effective approaches for studying SelU-tRNA interactions under high-pressure conditions?

Studying SelU-tRNA interactions under high pressure requires specialized techniques:

• High-pressure adapted biophysical methods:

  • HP-SAXS (High-Pressure Small-Angle X-ray Scattering):

    • Monitors protein-RNA complex formation in solution under pressure

    • Can detect pressure-induced conformational changes

  • HP-FRET (High-Pressure Förster Resonance Energy Transfer):

    • Label tRNA and SelU with fluorescent dyes

    • Measure changes in distance/orientation as a function of pressure

  • High-pressure NMR spectroscopy:

    • Analyze chemical shift perturbations upon complex formation

    • Monitor pressure effects on binding interfaces

• Biochemical approaches under pressure:

  • Filter-binding assays:

    • Use high-pressure vessels with sampling capacity

    • Determine dissociation constants at various pressures

  • Electrophoretic mobility shift assays (EMSA):

    • Pre-incubate components under pressure

    • Analyze complex formation after pressure release

• Computational methods:

  • Molecular dynamics simulations:

    • Model SelU-tRNA interactions under different pressure conditions

    • Identify key residues involved in pressure adaptation

  • QM/MM (Quantum Mechanics/Molecular Mechanics):

    • Study electronic changes in the active site under pressure

    • Model transition states during catalysis

• Genetic approaches:

  • Domain-swapping experiments:

    • Exchange domains between piezophilic and non-piezophilic SelU

    • Identify pressure-adaptive regions

  • Site-directed mutagenesis:

    • Target residues predicted to be involved in pressure adaptation

    • Assess impact on tRNA binding at different pressures

These approaches will provide insights into how P. profundum SelU maintains functional interactions with its tRNA substrates in the deep-sea environment.

• What are the challenges in developing high-throughput screening assays for SelU activity and how can they be overcome?

Developing high-throughput screening (HTS) assays for SelU activity faces several challenges:

Methodological solutions:

• Fluorescence-based assays:

  • Synthesize S2U-RNA containing fluorescent quencher

  • Design system where selenation alters fluorescence properties

  • Example: Position a quencher near S2U so that conformational change upon selenation relieves quenching

• Colorimetric selenium detection:

  • Adapt 3,3′-diaminobenzidine (DAB) reaction for selenium detection

  • Develop secondary reactions that produce colorimetric change upon selenation

• Coupled enzyme assays:

  • Link selenophosphate consumption to NAD(P)H oxidation

  • Monitor absorbance changes at 340 nm in real-time

• High-pressure microplate adaptations:

  • Design specialized pressure vessels for 96-well plate format

  • Incorporate optical windows for spectroscopic measurements

  • Implement automated pressure cycling for multiple condition testing

• Mass spectrometry-based screening:

  • Develop MALDI-TOF methods for rapid substrate/product identification

  • Implement automated sample handling and data analysis

These approaches would enable screening of enzyme variants, substrate specificity, and inhibitors while accounting for the unique challenges of working with a piezophilic enzyme.

• How can structural biology techniques be applied to understand pressure adaptation in P. profundum SelU?

Structural biology offers powerful approaches to understand pressure adaptation in P. profundum SelU:

• Comparative X-ray crystallography:

  • Crystallize P. profundum SelU and mesophilic homologs (e.g., E. coli SelU)

  • Identify structural differences in catalytic domains

  • Analyze cavities and packing density (typically reduced in pressure-adapted proteins)

  • Examine surface charge distribution and hydration patterns

• High-pressure X-ray crystallography:

  • Use diamond anvil cells to subject protein crystals to high pressure

  • Observe pressure-induced conformational changes

  • Identify regions of structural flexibility/rigidity

• Cryo-electron microscopy (cryo-EM):

  • Visualize SelU-tRNA complexes at near-atomic resolution

  • Compare binding interfaces under different pressure conditions

  • Observe conformational ensembles that may be important for function

• NMR spectroscopy:

  • Measure dynamics and flexibility at different pressures

  • Identify residues with unusual pressure responses

  • Study hydrogen/deuterium exchange rates as indicators of structural stability

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Probe protein dynamics and solvent accessibility

  • Compare exchange rates at atmospheric vs. high pressure

  • Identify regions with pressure-dependent flexibility

• Computational approaches:

  • Molecular dynamics simulations under pressure

  • Calculate volume changes associated with protein motions

  • Predict pressure-sensitive regions and interactions

Typical pressure adaptations in proteins from piezophiles include increased flexibility, reduced core hydrophobicity, and increased surface hydration. Structural studies would reveal which of these adaptations are employed by P. profundum SelU to maintain function in the deep sea, where pressures can reach 30 MPa or higher .

• What is known about the regulatory mechanisms controlling selU expression in P. profundum and how are they influenced by environmental factors?

The regulation of selU in P. profundum is not fully characterized, but insights can be drawn from what is known about pressure-responsive gene regulation in this organism:

• Pressure-responsive transcription:

  • P. profundum employs multiple pressure-responsive regulatory systems

  • The ToxR protein, a transmembrane DNA-binding regulator, influences gene expression in a pressure-dependent manner

  • Flagellar gene clusters in P. profundum show pressure-dependent expression patterns

• Temperature effects:

  • As a psychrophilic organism, P. profundum shows temperature-dependent gene expression

  • Stress response genes including htpG, dnaK, dnaJ, and groEL are upregulated in response to atmospheric pressure

  • SelU expression may be co-regulated with these stress response systems

• Selenium availability:

  • Selenium incorporation systems likely respond to selenium availability

  • SelD (selenophosphate synthetase) is often co-regulated with other selenoprotein machinery

• Growth phase-dependent regulation:

  • Many tRNA modification enzymes show growth phase-dependent expression

  • In P. profundum, solute distribution patterns change with growth stage

• Potential experimental approaches to study regulation:

  • RNA-seq analysis at different pressures, temperatures, and selenium concentrations

  • Promoter-reporter fusions to monitor selU expression under varying conditions

  • ChIP-seq to identify transcription factors binding to the selU promoter region

  • Proteomics to correlate SelU protein levels with environmental conditions

Understanding the regulatory mechanisms would provide insights into how P. profundum integrates selenium utilization with its adaptation to the deep-sea environment. The transcriptional landscape analysis of P. profundum revealed complex expression patterns with over 460 small RNA genes that could potentially play roles in regulating selenoprotein expression .

• How does the selenation activity of P. profundum SelU compare with homologs from non-piezophilic bacteria, and what does this reveal about deep-sea adaptation?

A comparative analysis of SelU from piezophilic and non-piezophilic bacteria could reveal key adaptations:

Expected differences in enzymatic properties:

Methodological approach for comparative analysis:

• Enzyme kinetics under pressure:

  • Measure reaction rates at pressures ranging from 0.1 to 50 MPa

  • Determine pressure optima and inhibitory pressures

  • Calculate activation volumes for both reaction steps

• Biophysical characterization:

  • Compare thermal and pressure stability profiles

  • Measure conformational changes using spectroscopic techniques

• Mutagenesis studies:

  • Identify residues unique to P. profundum SelU

  • Create chimeric enzymes with domains swapped between piezophilic and non-piezophilic SelU

  • Test activity under various pressure conditions

Similar comparative approaches with other enzymes from P. profundum have revealed remarkable adaptations. For example, flagellar motility in P. profundum SS9 actually increases at 30 MPa and maintains function up to 150 MPa, while E. coli motility is severely inhibited at elevated pressures . This suggests that P. profundum enzymes have evolved specific mechanisms to maintain or even enhance activity under deep-sea conditions.

• What are the implications of modified tRNAs containing Se2U for translation accuracy and efficiency in high-pressure environments?

The presence of Se2U modifications in tRNAs likely has significant impacts on translation in high-pressure environments:

• Codon-anticodon interactions:

  • Se2U in the wobble position affects base-pairing properties

  • Selenium has larger atomic radius than sulfur, potentially influencing steric interactions

  • May provide more stable or precise recognition under high pressure

• Ribosome dynamics:

  • High pressure can disturb ribosome structure and function

  • Modified tRNAs may help maintain proper ribosomal interactions

  • Could stabilize tRNA positioning in the A, P, and E sites under pressure

• Translation kinetics:

  • Se2U modifications may enhance translation rate or accuracy

  • Could compensate for pressure-induced changes in reaction rates

  • May be particularly important for pressure-specific gene expression

• Mistranslation prevention:

  • Se2U might reduce misincorporation of amino acids

  • Particularly important under stress conditions where translation fidelity is challenged

  • Could contribute to protein quality control at high pressure

• Proteome composition:

  • Impact on codon usage and amino acid incorporation patterns

  • May influence the abundance of pressure-regulated proteins

  • Could facilitate translation of genes needed for pressure adaptation

Experimental approaches to investigate these effects:

• In vitro translation assays:

  • Compare translation efficiency using Se2U-modified vs. unmodified tRNAs

  • Perform assays under varying pressure conditions

  • Measure misincorporation rates and elongation speeds

• Ribosome profiling:

  • Analyze ribosome occupancy and translation efficiency genome-wide

  • Compare wild-type P. profundum with selU mutants

  • Identify transcripts most affected by selenouridine modification

• Structural studies of the ribosome:

  • Cryo-EM of ribosomes with Se2U-tRNAs under different pressures

  • Examine conformational changes in the decoding center

Understanding these implications would provide insights into the broader role of RNA modifications in environmental adaptation and could inform synthetic biology approaches for engineering pressure-tolerant organisms.

• What strategies can be employed to isolate and characterize native tRNAs containing selenouridine modifications from P. profundum?

Isolating and characterizing selenouridine-modified tRNAs from P. profundum requires specialized approaches:

• Growth and cultivation strategy:

  • Grow P. profundum under optimal conditions (15°C, 28 MPa)

  • Use pressure vessels similar to those described for motility studies

  • Supplement media with selenium source (e.g., sodium selenite)

  • Harvest cells in late exponential phase for optimal tRNA yield

• tRNA isolation methods:

  • Total tRNA extraction:

    • Use acidic phenol extraction (pH 4.5-5.0) to selectively enrich tRNAs

    • Apply size-exclusion chromatography to separate tRNAs from other RNAs

  • Specific tRNA isolation:

    • Design complementary oligonucleotides to known selenouridine-containing tRNAs (tRNALys, tRNAGlu, tRNAGln)

    • Use oligonucleotide-directed affinity chromatography

    • Consider solid-phase DNA probes complementary to conserved tRNA regions

• Analytical techniques for modification identification:

  • Mass spectrometry:

    • LC-MS/MS analysis of nucleoside mixtures after complete digestion

    • RNase mapping with MALDI-TOF MS to determine modification positions

    • Quantify Se2U vs. S2U nucleoside ratios

  • Selenium-specific detection:

    • ICP-MS for sensitive selenium quantification

    • Radioactive ⁷⁵Se labeling for tracking selenium incorporation

  • Chromatographic separation:

    • HPLC with selenium-specific detection

    • 2D-TLC of nucleoside mixtures with specialized staining

• Functional characterization:

  • In vitro translation assays:

    • Compare translation efficiency and accuracy using purified native tRNAs

    • Test performance under different pressure conditions

  • tRNA charging assays:

    • Examine aminoacylation kinetics of Se2U-containing tRNAs

    • Compare with synthetic or recombinant tRNAs lacking the modification

This systematic approach would provide valuable insights into the natural abundance and distribution of selenouridine modifications in P. profundum tRNAs and their functional significance in high-pressure adaptation.