Recombinant Geobacter sulfurreducens 7-cyano-7-deazaguanine synthase (queC)

What is 7-cyano-7-deazaguanine synthase (queC) and what reaction does it catalyze?

7-Cyano-7-deazaguanine synthase (EC 6.3.4.20), encoded by the queC gene, is an enzyme that catalyzes the formation of a nitrile group from a carboxylic acid and ammonia, requiring ATP as an energy source. Specifically, it converts 7-carboxy-7-deazaguanine to 7-cyano-7-deazaguanine (preQ₀) in the queuosine biosynthetic pathway . The reaction generates AMP and pyrophosphate as co-products, indicating an ATP-consuming mechanism .

The catalytic reaction can be represented as:

7-carboxy-7-deazaguanine + ATP + NH₃ → 7-cyano-7-deazaguanine (preQ₀) + AMP + PPi

This nitrile-forming reaction represents a relatively unusual biochemical transformation in biological systems, making queC of particular interest from an enzymatic mechanism perspective.

How is queC activity typically assayed in laboratory settings?

The enzymatic activity of queC can be measured using several complementary techniques:

• HPLC-MS based assay: This newly developed method allows for direct detection and quantification of the reaction products. The assay monitors the disappearance of 7-carboxy-7-deazaguanine and the appearance of 7-cyano-7-deazaguanine (preQ₀) .

• ³¹P NMR analysis: This technique is used to track the formation of AMP and pyrophosphate, which are co-products of the reaction. It provides direct evidence of ATP consumption and confirms the reaction mechanism .

• Fluorescence-based thermal-shift assay: This technique helps determine the stability of the enzyme under various conditions and can be used to optimize buffer compositions and identify stabilizing ligands .

When designing an assay for queC activity, researchers should consider the enzyme's pH optimum (approximately 9.5 for the G. kaustophilus enzyme) and temperature optimum (around 60°C for thermostable variants) . Reaction components typically include the enzyme, substrate (7-carboxy-7-deazaguanine), ATP, and an ammonia source in an appropriate buffer system.

What are the optimal conditions for queC enzyme activity?

Based on studies with the related enzyme from Geobacter kaustophilus, queC demonstrates specific biochemical characteristics that likely apply to the G. sulfurreducens enzyme as well:

• pH optimum: The enzyme shows optimal activity at pH 9.5, indicating a preference for slightly alkaline conditions .

• Temperature optimum: The enzyme exhibits an apparent temperature optimum at 60°C, reflecting its thermostable nature .

• Thermal stability: Like many enzymes from thermophilic organisms, queC demonstrates significant thermostability, which can be advantageous for recombinant expression and in vitro applications .

• Substrate specificity: The enzyme displays strict substrate specificity for its natural substrate 7-carboxy-7-deazaguanine, suggesting a highly evolved active site architecture .

For optimal activity in experimental settings, researchers should maintain these conditions while ensuring sufficient ATP and ammonia availability, as these are required co-substrates for the nitrile-forming reaction.

What techniques are used to express recombinant queC from Geobacter sulfurreducens?

While the search results don't specifically detail expression methods for G. sulfurreducens queC, the following approaches are methodologically sound based on related work with similar enzymes:

• Heterologous expression system: The gene can be cloned into an expression vector such as pET-based systems and expressed in E. coli strains optimized for recombinant protein production (e.g., BL21(DE3)) .

• Protein purification: A common approach involves adding affinity tags (His-tag, GST, etc.) to facilitate purification using affinity chromatography. This is typically followed by size-exclusion chromatography to ensure high purity.

• Expression optimization: Parameters such as induction temperature, IPTG concentration, and duration of expression need optimization. For enzymes from Geobacter species, lower temperatures (15-25°C) during induction may improve soluble protein yield.

• Protein stability considerations: The addition of stabilizing agents such as glycerol (1-10%) to storage buffers can help maintain enzymatic activity during storage .

• Activity verification: Following purification, enzyme activity should be verified using the assays described in FAQ 1.2 to confirm that the recombinant protein is functional.

How does queC fit into the broader queuosine biosynthetic pathway?

The queC enzyme is a critical component in the multi-step biosynthetic pathway leading to queuosine (Q), a modified nucleoside found in the wobble position of certain tRNAs:

• Pathway position: QueC catalyzes the conversion of 7-carboxy-7-deazaguanine to 7-cyano-7-deazaguanine (preQ₀), an intermediate step in the queuosine biosynthetic pathway .

• Upstream processing: Prior to queC action, earlier enzymes in the pathway generate 7-carboxy-7-deazaguanine from GTP through a series of transformations.

• Downstream processing: After queC generates preQ₀, this intermediate undergoes reduction to form preQ₁, which is then incorporated into tRNA by the enzyme tRNA-guanine transglycosylase (Tgt) .

• Final modifications: Following incorporation into tRNA, preQ₁ is further modified by QueA to form epoxyqueuosine (oQ), which is then reduced to queuosine (Q) by QueG, an enzyme that requires cobalamin, dithionite, and methyl viologen for activity in vitro .

This pathway is illustrated by the following sequence:
GTP → 7-carboxy-7-deazaguanine → preQ₀ (via queC) → preQ₁ → tRNA-preQ₁ → tRNA-oQ → tRNA-Q

The pathway's complexity highlights queC's important role in enabling the subsequent tRNA modifications that impact translation fidelity and efficiency.

How does substrate specificity of G. sulfurreducens queC compare to homologs from other bacteria?

The queC enzyme from thermophilic bacteria such as G. kaustophilus demonstrates strict substrate specificity for 7-carboxy-7-deazaguanine . While direct comparative studies between G. sulfurreducens queC and other bacterial homologs are not explicitly detailed in the search results, several methodological approaches can address this question:

• Substrate screening: Testing the G. sulfurreducens enzyme with structural analogs of 7-carboxy-7-deazaguanine to determine the extent of substrate promiscuity. The enzyme from G. kaustophilus shows high specificity, suggesting evolutionary conservation of this trait across related enzymes .

• Kinetic parameter comparison: Determining Km and kcat values for the G. sulfurreducens enzyme with various substrates and comparing these to published values for homologs. Lower Km values for the natural substrate would confirm specificity.

• Structural analysis: Homology modeling of the G. sulfurreducens queC based on crystal structures of homologs can provide insights into conserved active site residues that determine substrate specificity.

• Mutagenesis studies: Targeted mutations of active site residues predicted to interact with the substrate can help identify determinants of specificity and potentially engineer enzymes with modified substrate preferences.

The high specificity observed in the G. kaustophilus enzyme suggests that queC enzymes from different bacterial species, including G. sulfurreducens, may have evolved to recognize their natural substrate with high precision, although variations in efficiency and secondary substrate utilization may exist.

What role does queC play in the survival and metabolism of Geobacter species in subsurface environments?

The role of queC in Geobacter survival in subsurface environments must be considered within the context of the organism's unique ecological niche:

• Translational efficiency: As part of the queuosine modification pathway, queC contributes to proper tRNA modification, which may optimize translational efficiency and accuracy under the stress conditions found in subsurface environments .

• Stress adaptation: Geobacter species dominate in anoxic subsurface environments where Fe(III) reduction is the primary electron-accepting process . Proper tRNA modification may contribute to stress response and adaptation in these challenging environments.

• Integration with metal reduction: Genome-scale modeling of Geobacter in uranium-contaminated aquifers has shown that the organism's metabolic flux distribution changes significantly under different environmental conditions . The queuosine modification pathway, including queC, may influence translational regulation of key proteins involved in metal reduction.

• Competition dynamics: In subsurface environments, Geobacter competes with other organisms such as Rhodoferax for resources like acetate . Efficient translation through proper tRNA modification could provide a competitive advantage under resource-limited conditions.

• Nitrogen limitation response: In environments with limited ammonia, Geobacter relies on nitrogen fixation, which diverts carbon and electron flux toward respiration rather than biomass formation . Given queC's requirement for ammonia as a substrate, its activity might be regulated in response to nitrogen availability.

The connection between queC function and Geobacter's ecological success highlights the potential importance of tRNA modification in environmental adaptation, though direct experimental evidence linking queC to specific adaptive advantages in subsurface environments would require further investigation.

How can genome-scale metabolic modeling incorporate queC function in predicting Geobacter behavior during bioremediation?

Integrating queC function into genome-scale metabolic models of Geobacter species could enhance predictions of bioremediation outcomes through several approaches:

• Pathway incorporation: Adding the queuosine biosynthetic pathway, including queC reactions, to existing genome-scale models of G. sulfurreducens. This requires defining stoichiometry, reaction constraints, and gene-protein-reaction associations .

• Conditional expression modeling: Using transcriptomic data to predict queC expression levels under different environmental conditions, such as varying ammonium availability or acetate flux .

• Linking to translation efficiency: Developing sub-models that connect tRNA modification status to translation efficiency of key proteins involved in extracellular electron transfer and metal reduction .

• Integration with community models: Extending single-organism models to community models that predict interactions between Geobacter and competing organisms like Rhodoferax under different geochemical conditions .

• Validation with field data: Comparing model predictions with proteomics data from field samples, such as those from uranium-contaminated aquifers, to refine model parameters and improve prediction accuracy .

For example, genome-scale modeling has successfully predicted how Geobacter outcompetes Rhodoferax during bioremediation with high acetate concentrations, partly due to Geobacter's ability to fix nitrogen . Adding queC functionality could provide additional insights into how translational regulation contributes to this competitive advantage.

What methodological approaches can be used to study the in vivo regulation of queC expression in G. sulfurreducens?

Several complementary methodological approaches can be employed to understand the regulation of queC expression in G. sulfurreducens under environmentally relevant conditions:

• Transcriptomic analysis: RNA-Seq can be used to quantify queC mRNA levels under various growth conditions, such as different electron acceptors (fumarate vs. Fe(III) oxide) or electron donors (acetate vs. lactate) .

• Proteomics: Quantitative proteomics can determine QueC protein abundance across different conditions, as has been done for other Geobacter proteins across laboratory and field experiments .

• Reporter gene fusions: Constructing fusions between the queC promoter and reporter genes (e.g., luciferase) can enable real-time monitoring of gene expression in response to environmental changes.

• In situ gene expression: For field studies, primers targeting the rpsC gene can be used to estimate in situ growth rates of Geobacter species, providing context for queC expression data .

• Promoter analysis: Identifying transcription factor binding sites in the queC promoter region can help elucidate regulatory pathways controlling its expression. The search results mention GSU0514, a putative transcriptional regulator in G. sulfurreducens that was modified during laboratory evolution experiments .

• ChIP-Seq analysis: This technique can identify transcription factors that bind to the queC promoter region in vivo under different growth conditions.

By applying these approaches, researchers can determine how queC expression is regulated in response to environmental factors relevant to bioremediation, such as metal availability, carbon source, and nitrogen limitation.

How do post-translational modifications affect the activity of recombinant G. sulfurreducens queC?

While the search results don't provide specific information about post-translational modifications (PTMs) of G. sulfurreducens queC, investigating their potential impact involves several methodological approaches:

• Mass spectrometry analysis: High-resolution MS can identify PTMs such as phosphorylation, acetylation, or methylation in purified recombinant queC. Comparing PTM patterns between recombinant and native enzyme can reveal differences that might affect activity.

• Site-directed mutagenesis: Modifying potential PTM sites (e.g., changing phosphorylatable serine residues to alanine) can help determine their functional significance by comparing the activity of mutant enzymes to wild-type queC.

• In vitro modification: Treating purified recombinant queC with kinases, acetylases, or other modifying enzymes in vitro can assess how specific PTMs affect enzymatic activity, stability, or substrate binding.

• Activity assays under varying conditions: Testing enzyme activity across conditions that might influence PTM status (e.g., different redox states) could reveal regulatory mechanisms.

• Structural analysis: Molecular modeling and, ideally, crystal structures of queC with and without specific PTMs can provide insights into how modifications affect enzyme conformation and active site architecture.

For recombinant expression, it's important to consider that E. coli or other heterologous hosts may not reproduce the same PTM pattern as native G. sulfurreducens, potentially leading to differences in enzyme properties. Comparative studies between native and recombinant enzymes would be valuable to identify such differences.

What are the key considerations for optimizing recombinant expression of G. sulfurreducens queC?

Optimizing recombinant expression of G. sulfurreducens queC requires addressing several factors:

• Expression system selection: While E. coli is a common host, its cytoplasmic redox environment and chaperone systems differ from Geobacter. Consider testing multiple E. coli strains (BL21(DE3), Rosetta, etc.) or alternative hosts more similar to Geobacter.

• Codon optimization: Adapting the G. sulfurreducens queC gene sequence to the codon usage bias of the expression host may improve translation efficiency and yield.

• Protein solubility: Fusion tags (MBP, SUMO, etc.) can enhance solubility. Testing expression at lower temperatures (15-20°C) may also reduce inclusion body formation.

• Metal cofactor incorporation: If queC requires metal cofactors for proper folding or activity, supplementing the growth medium with appropriate metals may be necessary. For example, if the enzyme contains iron-sulfur clusters like some related Geobacter proteins, iron supplementation might be beneficial .

• Induction parameters: Systematically testing induction conditions (IPTG concentration, cell density at induction, duration) can maximize active protein yield.

• Protein purification strategy: Design a purification scheme that preserves enzyme activity. For thermostable enzymes like queC, a heat treatment step might help remove contaminating host proteins .

• Activity verification: Develop a high-throughput assay to rapidly test activity of different expression constructs and purification fractions.

Implementing a factorial experimental design to simultaneously test multiple parameters can efficiently identify optimal expression conditions.

How can isotope labeling approaches be used to investigate the catalytic mechanism of queC?

Isotope labeling provides powerful tools for elucidating the catalytic mechanism of queC:

• ¹⁸O labeling of substrate carboxyl group: Using 7-carboxy-7-deazaguanine with an ¹⁸O-labeled carboxyl group would allow tracking of oxygen fate during the reaction. If ¹⁸O appears in released products (e.g., pyrophosphate), it would provide evidence for specific reaction intermediates.

• ¹⁵N labeling of ammonia: Using ¹⁵NH₃ as a substrate would confirm ammonia as the direct nitrogen source for the nitrile group in the product. Mass spectrometry analysis would show ¹⁵N incorporation into the 7-cyano-7-deazaguanine product.

• ³¹P and ¹⁷O labeling in ATP: Using ATP with specific phosphate oxygens labeled with ¹⁷O can help determine the reaction mechanism by tracking which oxygens end up in AMP versus pyrophosphate .

• Kinetic isotope effects: Comparing reaction rates with normal substrates versus isotopically labeled substrates can identify rate-limiting steps in the catalytic mechanism.

• NMR spectroscopy: Using ¹³C or ¹⁵N labeled substrates enables real-time NMR monitoring of the reaction, potentially capturing transient intermediates.

These approaches could help answer key mechanistic questions: Does ATP directly activate the carboxyl group? Is ammonia directly incorporated, or is the nitrogen derived from an amino acid or other nitrogen donor? How does the enzyme catalyze the challenging conversion of a carboxyl to a nitrile group?

What techniques can be used to determine the crystal structure of G. sulfurreducens queC?

Determining the crystal structure of G. sulfurreducens queC would provide valuable insights into its catalytic mechanism. The following methodological approach is recommended:

• Protein production optimization:

  • Express queC with removable affinity tags for purification

  • Ensure high purity (>95%) by multi-step chromatography

  • Verify protein homogeneity by dynamic light scattering

  • Test thermal stability using differential scanning fluorimetry to identify stabilizing buffer conditions

• Crystallization screening:

  • Perform initial screens using commercial sparse matrix kits

  • Test crystallization with various ligands (substrate, product, ATP analogs)

  • Optimize promising conditions by varying precipitant concentration, pH, and additives

  • Consider crystallization at different temperatures (4°C vs. room temperature)

• Structure determination:

  • Collect X-ray diffraction data at synchrotron radiation facilities

  • For phase determination, consider:
    a. Molecular replacement using structures of homologous proteins
    b. Experimental phasing using selenomethionine-labeled protein
    c. Heavy atom soaking for isomorphous replacement

• Structure analysis:

  • Identify the active site and substrate binding pocket

  • Analyze potential catalytic residues

  • Compare with structures of other nitrile-forming enzymes

  • Model substrate binding and enzyme mechanism

• Validation studies:

  • Confirm importance of identified residues through site-directed mutagenesis

  • Test catalytic activity of mutants using established assays

This approach has been successful for determining structures of related enzymes and would provide critical insights into the unique nitrile-forming chemistry of queC.

How might queC be engineered for improved catalytic efficiency or altered substrate specificity?

Engineering queC for enhanced properties could follow several strategic approaches:

• Structure-guided mutagenesis: Once the structure is known, rational design can target:

  • Active site residues to enhance substrate binding (lower Km)

  • Catalytic residues to improve turnover rate (higher kcat)

  • Substrate recognition elements to alter specificity

• Directed evolution: Creating libraries of queC variants through:

  • Error-prone PCR to generate random mutations

  • DNA shuffling with homologous queC genes from different organisms

  • Site-saturation mutagenesis at key positions
    Selection methods would need a high-throughput assay to identify improved variants.

• Computational design: Using algorithms to predict beneficial mutations:

  • Molecular dynamics simulations to identify rate-limiting conformational changes

  • Quantum mechanical/molecular mechanical (QM/MM) calculations to optimize transition state interactions

  • Sequence-based machine learning approaches trained on diverse queC homologs

• Domain swapping: Creating chimeric enzymes with domains from thermophilic homologs like those from G. kaustophilus to enhance stability while maintaining the substrate specificity of G. sulfurreducens queC .

• Adaptation to environmental conditions: Laboratory evolution experiments similar to those performed for lactate metabolism in G. sulfurreducens could yield queC variants with improved performance under specific conditions .

Success metrics would include increased kcat/Km ratios, expanded substrate range, or enhanced stability under conditions relevant to bioremediation applications.

What is the potential role of queC in engineering Geobacter strains for enhanced bioremediation capabilities?

Leveraging queC for enhanced bioremediation with engineered Geobacter strains presents several promising approaches:

• Optimizing translation efficiency: Since queC contributes to tRNA modification, engineering its expression could enhance translation of key proteins involved in metal reduction and contaminant transformation .

• Adaptation to environmental stressors: Adjusting queC expression or properties could help Geobacter tolerate challenging conditions at contaminated sites, such as:

  • High metal concentrations

  • Fluctuating redox conditions

  • Nutrient limitations

  • Competing microorganisms

• Integration with genome-scale metabolic engineering: Using models that predict how queC and the queuosine pathway interact with central metabolism to identify targets for strain improvement. For example, models have already predicted how nitrogen limitation affects carbon flux distribution in Geobacter .

• Cross-species comparative analysis: Examining queC from Geobacter species with superior remediation capabilities to identify beneficial variants that could be introduced into G. sulfurreducens .

• Field testing protocol development: Creating standardized methods to monitor the performance of engineered strains in real bioremediation settings, including:

  • Quantifying queC expression using RT-PCR

  • Tracking growth rates with markers like rpsC

  • Monitoring metal reduction rates and contaminant transformation

The metabolic versatility of Geobacter species, including their ability to reduce uranium and compete effectively in subsurface environments when provided with appropriate electron donors like acetate, makes them promising candidates for engineered bioremediation applications .

How does queC from G. sulfurreducens compare to other enzymes involved in tRNA modification pathways?

Comparing queC to other tRNA modification enzymes reveals important distinctions in mechanism and function:

• Mechanistic uniqueness: Unlike most tRNA modification enzymes that perform relatively common chemical transformations (methylation, thiolation, etc.), queC catalyzes an unusual nitrile formation reaction. This reaction requires ATP activation and represents a distinctive mechanistic class .

• Substrate interactions:

  • QueC acts on a free nucleobase (7-carboxy-7-deazaguanine), not on tRNA-bound substrates

  • In contrast, enzymes like QueA modify nucleosides already incorporated into tRNA

  • Tgt exchanges a whole nucleobase in the tRNA rather than modifying an existing one

• Pathway position and energetics:

  • QueC functions early in the queuosine pathway, before nucleobase incorporation into tRNA

  • Unlike many tRNA modifications that require only cofactors, queC consumes ATP, making it energetically costly

  • The later pathway enzyme QueG requires unusual cofactors (cobalamin) and reducing agents (dithionite, methyl viologen)

• Evolutionary conservation:

  • The strict substrate specificity of queC suggests strong evolutionary conservation

  • Some tRNA modification pathways show more variability across bacterial species

• Regulation patterns:

  • Unlike some tRNA modification enzymes that are constitutively expressed, queC expression may respond to environmental conditions based on patterns observed for other Geobacter genes

This comparison highlights queC's distinctive role in the elaborate pathway of tRNA modification, which ultimately contributes to translational fidelity and efficiency.

What are the key differences between recombinant expression and native expression of queC in Geobacter sulfurreducens?

Several important differences may exist between recombinantly expressed and native queC:

• Protein folding environment:

  • Heterologous expression in E. coli occurs in a cytoplasmic environment with different pH, redox potential, and ion concentrations than in Geobacter

  • Native expression benefits from co-evolved chaperone systems specific to Geobacter

• Post-translational modifications:

  • Recombinant expression may lack Geobacter-specific PTMs that could affect activity

  • Differences in phosphorylation, acetylation, or other modifications might alter enzyme properties

• Metal content and cofactors:

  • If queC requires specific metal ions or cofactors, their availability may differ between expression systems

  • Geobacter's metal-rich environment might provide optimal cofactor loading during native expression

• Protein-protein interactions:

  • In its native environment, queC may interact with other enzymes in the queuosine pathway

  • These interactions could affect activity, stability, or localization in ways not reproduced in recombinant systems

• Regulatory context:

  • Native expression responds to environmental cues relevant to Geobacter's metabolism

  • Laboratory evolution has shown that transcriptional regulators in Geobacter can adapt to new selective pressures

  • Recombinant expression typically uses constitutive or inducible promoters disconnected from natural regulation

Understanding these differences is critical for interpreting results from recombinant enzyme studies and applying them to understand the enzyme's physiological role in Geobacter.

How does the ATP utilization mechanism of queC compare to other ATP-dependent enzymes?

The ATP utilization mechanism of queC can be compared to other ATP-dependent enzymes to highlight its distinctive features:

• ATP consumption pattern:

  • QueC generates AMP and pyrophosphate as co-products, indicating ATP is cleaved after the α-β phosphodiester bond

  • This pattern is shared with some other carboxyl-activating enzymes like aminoacyl-tRNA synthetases and fatty acid CoA ligases

  • In contrast, kinases cleave ATP after the β-γ bond, producing ADP

• Activation mechanism:

  • QueC likely activates the carboxyl group of 7-carboxy-7-deazaguanine, forming an acyl-adenylate intermediate

  • This differs from kinases, which transfer the γ-phosphate to substrate hydroxyl or amino groups

  • The subsequent conversion to a nitrile is unusual and may involve a unique catalytic mechanism

• Structural features:

  • ATP-binding domains in queC likely include a P-loop or Walker A motif for phosphate binding

  • The enzyme must coordinate both ATP and carboxylate substrate binding

  • Structural features that facilitate ammonia incorporation and water elimination would be unique to queC

• Energy efficiency:

  • The ATP consumption by queC (one ATP per reaction) represents a significant energy investment

  • This suggests the nitrile formation is challenging enough to warrant ATP expenditure

  • Some other modifications achieve similar chemical complexity without ATP hydrolysis

• Evolutionary context:

  • The conservation of this ATP-dependent mechanism across bacterial species suggests its importance

  • The queC reaction may represent an evolutionary solution to the challenge of creating a nitrile group in biological systems

These comparisons place queC within the broader context of ATP-utilizing enzymes while highlighting its specialized role in queuosine biosynthesis.