Recombinant Photobacterium profundum tRNA-modifying protein ygfZ (PBPRA3100)

What is Photobacterium profundum YgfZ and how does it function in tRNA modification pathways?

P. profundum YgfZ belongs to the COG0354 protein family of folate-binding proteins involved in tRNA modification and iron-sulfur (Fe-S) cluster metabolism. Similar to its E. coli homolog, P. profundum YgfZ likely participates in maintaining the activity of Fe-S enzymes like MiaB, which catalyzes the methylthiolation of tRNAs . The protein is part of a complex regulatory network that influences tRNA modification, particularly in the formation of 2-methylthio N(6)-isopentenyladenosine in some tRNAs . As P. profundum is a deep-sea psychrohalophile, its YgfZ has likely evolved specific adaptations for functioning under high pressure and low temperature conditions.

P. profundum strain SS9 has been adopted as a model for piezophily (pressure adaptation), with its genome sequence (6.4 megabase pairs) revealing high metabolic versatility for life in deep-sea environments . The YgfZ protein in this organism represents an important component of its adaptation to extreme conditions.

How does the structure of P. profundum YgfZ relate to its function in Fe-S cluster metabolism?

While a specific crystal structure of P. profundum YgfZ has not been reported in the provided literature, structural information can be inferred from homologs. The E. coli YgfZ structure provides insights into the folate-binding domain that is likely conserved in P. profundum. The folate-binding capability is crucial for its function, as YgfZ paralogs mediate folate-dependent formaldehyde removal .

The protein likely contains a conserved structural core that enables folate-binding and plays a role in preventing damage to Fe-S enzymes or repairing damaged Fe-S clusters. Given P. profundum's adaptation to deep-sea environments, structural modifications may exist that enhance protein stability and activity under high pressure conditions.

What expression systems are optimal for producing recombinant P. profundum YgfZ?

Based on established protocols for other P. profundum proteins, E. coli is a suitable expression system for recombinant production. When designing an expression system:

• Vector selection: Use a vector with a strong promoter (T7, tac) and add a purification tag (His, GST) to facilitate downstream purification.

• Expression conditions: Optimize for lower temperatures (15-20°C) to mimic the psychrophilic nature of P. profundum and improve protein solubility.

• Codon optimization: Consider codon optimization for E. coli expression, as P. profundum may have different codon usage patterns .

The expression of recombinant P. profundum proteins in E. coli has been demonstrated with other proteins like putative phosphotransferase (PBPRA1750), showing this approach is viable .

What purification strategies yield high-quality P. profundum YgfZ for functional studies?

For optimal purification of recombinant P. profundum YgfZ:

• Initial clarification: After cell lysis, perform centrifugation at 10,000×g at 4°C to remove cellular debris .

• Affinity chromatography: Use immobilized metal affinity chromatography (IMAC) if a His-tag is incorporated.

• Size exclusion chromatography: Separate monomers from potential dimers, as observed with other P. profundum proteins .

• Storage conditions: Store with 50% glycerol at -20°C/-80°C to maintain stability, with an expected shelf life of 12 months for lyophilized protein .

For functional studies, include folate cofactors during purification and storage to maintain the protein's activity. The solubility and stability of the protein can be improved by adding stabilizing agents appropriate for psychrophilic proteins.

How can researchers assess the in vivo activity of P. profundum YgfZ?

To evaluate YgfZ activity in vivo, several approaches can be employed:

• Genetic complementation: Test whether P. profundum YgfZ can complement an E. coli ygfZ deletion, particularly under stress conditions .

• tRNA modification analysis: Measure the levels of modified tRNA nucleosides using LC-MS/MS:

  • Extract total RNA from wild-type and ΔygfZ strains

  • Enzymatically digest RNA to nucleosides

  • Separate and detect modified nucleosides using UPLC coupled to mass spectrometry

• Fe-S enzyme activity assays: Monitor the activity of Fe-S enzymes like MiaB or RimO:

  Enzyme	WT activity	ΔygfZ activity	Assay method
  RimO	40%	0.03%	LC-MS² of S12 thiomethylation
  MiaB	30%	2%	Analysis of tRNA modification

  Table adapted from complementation data in reference

• Growth phenotyping: Compare growth rates at different temperatures and pressures, as ygfZ mutants often show temperature-dependent phenotypes .

How does YgfZ interact with other tRNA modification enzymes in P. profundum?

YgfZ functions within a complex network of tRNA modification enzymes. Based on studies in E. coli and other bacteria, P. profundum YgfZ likely:

• Supports MiaB activity: Helps maintain the activity of MiaB, which catalyzes the methylthiolation of some tRNAs .

• Counters MnmEG effects: Based on E. coli studies, YgfZ appears to counteract the folate-dependent action of MnmEG, which may damage MiaB and other Fe-S enzymes .

• Participates in regulatory networks: YgfZ influences tRNA modification through a network that may involve other factors specific to deep-sea adaptation.

The interaction network is illustrated by evidence showing that deleting mnmE can suppress phenotypes associated with ygfZ deletion, indicating an opposing relationship between these tRNA modification pathways .

What synergistic effects occur when YgfZ is combined with other tRNA modification defects?

Synthetic lethal interactions involving tRNA modification genes provide insights into functional relationships. Recent research has employed pairwise deletion matrices to identify such interactions:

• Condition-dependent synthetic lethality: YgfZ may exhibit synthetic lethal interactions with other tRNA modification genes under specific environmental conditions .

• Growth rate effects: Double deletions involving ygfZ and other tRNA modification genes often result in more severe phenotypes than single deletions .

• Pressure-dependent effects: In P. profundum, these interactions may be particularly important under high-pressure conditions where tRNA modifications play critical roles in maintaining translation efficiency.

The search results indicate that "deletion of rlmN, mnmA, trmJ or truA resulted in synthetic lethal combinations with several other tRNA modification mutants" , suggesting potential interactions with YgfZ in similar networks.

How does P. profundum YgfZ contribute to deep-sea environmental adaptation?

P. profundum strain SS9 is a model piezophile (pressure-loving organism) that grows optimally at 28 MPa and 15°C . YgfZ likely contributes to deep-sea adaptation through:

• Maintaining tRNA modification integrity: Ensuring proper tRNA modifications that support translation under high-pressure conditions.

• Fe-S enzyme protection: Protecting crucial Fe-S enzymes from pressure-induced damage or instability.

• Integration with pressure-responsive systems: YgfZ may interact with other pressure-responsive gene regulatory systems, like ToxR, which regulates gene expression in response to pressure in P. profundum .

A proteomic analysis of P. profundum grown under different pressure conditions revealed differential expression of proteins involved in various metabolic pathways . While YgfZ was not specifically mentioned, proteins involved in similar pathways showed pressure-dependent regulation.

What structural features distinguish P. profundum YgfZ from mesophilic homologs?

While specific structural data for P. profundum YgfZ is not provided in the search results, psychrophilic proteins typically exhibit adaptations that can be inferred:

• Increased flexibility: Psychrophilic proteins often have fewer rigid structural elements to maintain activity at low temperatures.

• Surface charge modifications: Altered surface charge distribution to maintain solubility and interactions under high pressure conditions.

• Folate binding adaptations: The folate-binding domain may have specific adaptations to function efficiently under deep-sea conditions of high pressure and low temperature.

The crystal structure of a psychrohalophilic α-carbonic anhydrase from P. profundum reveals a unique dimer interface with a chloride ion not observed in other homologs . Similar unique features may exist in P. profundum YgfZ.

How can P. profundum YgfZ be used as a model to understand tRNA modification in extremophiles?

P. profundum YgfZ offers a valuable model for understanding tRNA modification mechanisms in extremophiles:

• Comparative studies: Compare the activity and substrate specificity of P. profundum YgfZ with mesophilic counterparts to identify pressure-adaptive features.

• In vitro reconstitution: Reconstitute tRNA modification systems including YgfZ from P. profundum to examine how they function under varying pressure conditions.

• Evolutionary analysis: Use YgfZ sequences from piezophiles, psychrophiles, and mesophiles to trace the evolutionary adaptations to extreme environments.

Recent research has shown that "tRNA modifications are master regulators of codon usage and optimality in various tissues" , suggesting similar regulatory roles may exist in bacteria adapted to extreme environments.

What insights can P. profundum YgfZ provide about the evolution of tRNA modification systems?

P. profundum YgfZ can illuminate evolutionary aspects of tRNA modification:

• Niche-specific adaptations: Comparison with YgfZ from non-extremophiles reveals adaptations specific to high-pressure environments.

• Conservation patterns: Analysis of conserved residues across diverse species helps identify functionally critical regions versus adaptable regions.

• Co-evolution with partner proteins: Examining how YgfZ co-evolved with interacting partners like MiaB provides insights into the evolution of tRNA modification networks.

The search results indicate that YgfZ "occurs in many genomes that lack MnmEG (e.g., Actinobacteria), implying that MiaB is subject to additional types of damage" . This suggests functional versatility across different evolutionary lineages.

How does YgfZ's folate-binding mechanism relate to its function in tRNA modification?

The folate-binding capacity of YgfZ is central to its function:

• Formaldehyde transfer: YgfZ may use folate to capture formaldehyde units that could damage Fe-S clusters .

• Repair mechanism: Evidence suggests YgfZ might reverse erroneous formaldehyde transfers mediated by MnmEG to proteins like MiaB:

  "It is conceivable that MnmEG occasionally and by mistake transfers a formaldehyde unit to a sensitive residue in MiaB and, perhaps, to such residues in other proteins. YgfZ might strip this unit off and transfer it to THF."

• Pressure effects on binding: High pressure may influence folate binding and the subsequent chemical reactions, necessitating adaptations in the P. profundum YgfZ.

Experiments investigating these mechanisms could employ isotope-labeled folate derivatives and mass spectrometry to trace formaldehyde transfer paths in the presence and absence of YgfZ.

What are common challenges in working with recombinant P. profundum YgfZ and how can they be addressed?

Researchers working with P. profundum YgfZ may encounter several challenges:

• Low expression yields: Optimize by:

  • Lowering expression temperature to 15°C

  • Using a host strain with rare codons (e.g., Rosetta)

  • Testing different induction conditions (IPTG concentration, induction time)

• Protein instability: Address by:

  • Including folate derivatives during purification

  • Adding stabilizing agents like glycerol (50% final concentration)

  • Avoiding repeated freeze-thaw cycles

• Activity loss during purification: Mitigate by:

  • Maintaining reducing conditions throughout purification

  • Including folate cofactors in all buffers

  • Purifying under anaerobic conditions if possible to preserve Fe-S cluster integrity

How can researchers design experiments to differentiate between direct and indirect effects of YgfZ on tRNA modification?

To distinguish direct from indirect YgfZ effects:

• In vitro reconstitution experiments:

  • Purify YgfZ and potential target proteins (e.g., MiaB)

  • Test direct interactions using techniques like surface plasmon resonance

  • Perform in vitro activity assays with purified components

• Genetic suppressor screens:

  • Identify suppressors of ygfZ deletion phenotypes

  • Analyze whether suppressors act in the same or parallel pathways

• Targeted mutagenesis:

  • Create point mutations in specific YgfZ domains

  • Assess which functions are affected by each mutation

• Time-resolved experiments:

  • Monitor changes in tRNA modifications immediately after YgfZ inactivation

  • Direct effects will manifest faster than indirect ones

The complexity of tRNA modification networks requires careful experimental design to deconvolute direct and indirect effects, as illustrated by studies showing that "MnmEG activation or repression cannot be used to predict the influence of pressure on gene expression" .