Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni 7-cyano-7-deazaguanine synthase (queC)

What is 7-cyano-7-deazaguanine synthase (queC) and what role does it play in Leptospira interrogans?

7-Cyano-7-deazaguanine synthase (EC 6.3.4.20) is an enzyme that catalyzes the formation of a nitrile from a carboxylic acid and ammonia at the expense of ATP, specifically converting 7-carboxy-7-deazaguanine to 7-cyano-7-deazaguanine (preQ0) . In Leptospira interrogans, queC is part of the queuosine biosynthetic pathway, which is essential for tRNA modification. This enzyme represents an important component in bacterial metabolism and potentially impacts pathogenesis through its role in translational fidelity.

How does queC from Leptospira interrogans compare to homologs in other bacterial species?

While specific comparative data for Leptospira queC is limited, queC from other thermophilic bacteria like G. kaustophilus demonstrates high thermostability, a pH optimum of approximately 9.5, and an apparent temperature optimum around 60°C . Leptospira queC likely shares core catalytic mechanisms but may exhibit different biochemical characteristics adapted to the environmental conditions Leptospira encounters during its complex lifecycle. Sequence alignment analysis typically reveals conserved catalytic domains across bacterial species with adaptations in non-catalytic regions.

What are the specific biochemical reaction parameters for queC in Leptospira?

queC catalyzes the ATP-dependent conversion of 7-carboxy-7-deazaguanine to preQ0, releasing AMP and pyrophosphate as co-products . While specific kinetic parameters for Leptospira queC haven't been fully characterized, similar enzymes show strict substrate specificity for 7-carboxy-7-deazaguanine. The reaction involves ATP hydrolysis coupled to the amidation and subsequent dehydration to form the nitrile group. Experimental determination of Km, Vmax, and catalytic efficiency would require recombinant expression and biochemical assays under varying substrate concentrations and environmental conditions.

What expression systems are most effective for recombinant production of Leptospira queC?

For recombinant Leptospira proteins, E. coli-based expression systems have proven effective, as demonstrated with other Leptospira proteins . For queC specifically, consider using the following approach:

• Codon-optimized synthetic gene construct

• Expression vector with T7 promoter system (pET series)

• BL21(DE3) E. coli strain or derivatives

• N-terminal His-tag fusion for purification

• IPTG-inducible expression system

Recent advances in Leptospira genetics now allow for homologous expression using IPTG-inducible systems, which may provide proteins with native post-translational modifications . The choice between heterologous (E. coli) and homologous (Leptospira) expression depends on research goals and required protein authenticity.

What purification strategy yields the highest purity and activity for recombinant queC?

A multi-step purification strategy is recommended:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA for initial capture of His-tagged queC

• Size-exclusion chromatography to remove aggregates and impurities

• Ion-exchange chromatography for final polishing

Storage recommendations include lyophilization or storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0 with addition of 5-50% glycerol for long-term storage at -20°C/-80°C . Avoid repeated freeze-thaw cycles as this may compromise enzyme activity.

How can I assess the structural integrity and activity of purified queC?

Multiple complementary approaches should be used:

• Structural integrity assessment:

  • SDS-PAGE (expect >90% purity)

  • Circular dichroism spectroscopy to assess secondary structure

  • Thermal shift assays to determine protein stability

  • Limited proteolysis to assess proper folding

• Activity assessment:

  • HPLC-MS based assay to detect conversion of 7-carboxy-7-deazaguanine to preQ0

  • 31P NMR to monitor ATP hydrolysis and pyrophosphate formation

  • Coupled enzyme assays measuring AMP production

Enzyme kinetics should be determined under optimal conditions (likely pH ~9.5, temperature ~60°C based on homologous enzymes) .

What structural features are important for queC function in Leptospira?

While specific structural data for Leptospira queC is not available, inference from homologous proteins suggests:

• ATP-binding domain characterized by Walker A and B motifs

• Substrate-binding pocket with high specificity for 7-carboxy-7-deazaguanine

• Catalytic residues mediating nitrile formation

• Possible oligomerization interfaces

Structural prediction using AlphaFold or RoseTTAFold may provide preliminary structural insights pending experimental determination. Site-directed mutagenesis of predicted catalytic residues would help identify essential structural elements.

How does the pH and temperature profile of Leptospira queC influence experimental design?

Based on homologous queC enzymes, Leptospira queC likely exhibits:

• pH optimum around 9.5

• Temperature optimum potentially lower than thermophilic homologs (~60°C)

These parameters significantly impact experimental design:

• Buffer selection: Bicine or CHES buffers for high pH conditions

• Temperature control: Precise regulation during kinetic assays

• Protein stability considerations: Addition of stabilizing agents for long-term storage

• Assay development: pH-sensitive detection methods require careful calibration

A comprehensive pH-activity and temperature-activity profile should be established to determine optimal conditions specific to Leptospira queC.

What are the substrate specificity determinants of queC from Leptospira interrogans?

queC enzymes typically demonstrate strict substrate specificity for 7-carboxy-7-deazaguanine . To investigate specificity determinants:

• Test substrate analogs with modifications to the carboxylic acid position, purine ring, or substituents

• Analyze binding pocket residues through homology modeling and site-directed mutagenesis

• Conduct saturation mutagenesis of residues lining the active site

• Utilize isothermal titration calorimetry (ITC) to measure binding affinities of substrates and analogs

Engineering altered specificity could lead to novel biotechnological applications or inhibitor development.

How can CRISPR/Cas9 technology be applied to study queC function in Leptospira?

Recent advances in Leptospira genetics allow for powerful CRISPR/Cas9-based approaches:

• Gene knockout: IPTG-inducible Cas9 expression combined with constitutive non-homologous end-joining (NHEJ) system facilitates queC knockout generation

• Gene knockdown: IPTG-induced dead Cas9 (dCas9) expression enables validation of gene essentiality without complete disruption

• Genome editing: Precise modifications to the queC gene to study structure-function relationships

• Transcriptional regulation: Monitoring expression under various environmental conditions

These approaches can be implemented using recently developed IPTG-inducible heterologous protein expression systems in Leptospira, significantly expanding the genetic toolkit for studying this bacterium .

What is the role of queC in Leptospira pathogenesis and how can this be investigated?

The connection between tRNA modification and bacterial pathogenesis is an emerging area of research. To investigate queC's role in Leptospira pathogenesis:

• Compare queC expression levels between pathogenic and non-pathogenic Leptospira species

• Construct conditional queC mutants and assess virulence in animal models

• Identify transcriptional and translational effects of queC deficiency using RNA-seq and proteomics

• Evaluate tRNA modification profiles and translational fidelity in queC mutants

• Assess stress response and adaptation to host environments in queC-deficient strains

This research could provide insights into novel virulence mechanisms and potential therapeutic targets for leptospirosis, a disease responsible for over 1 million human cases annually .

How might queC inhibitors be developed as potential therapeutics against leptospirosis?

Development of queC inhibitors could follow this strategic pathway:

• High-throughput screening of compound libraries against recombinant Leptospira queC

• Structure-based drug design using computational modeling

• Fragment-based drug discovery focusing on the ATP binding site

• Natural product screening for queC inhibitors

• Validation in cellular systems and animal models

Given the annual impact of over 1 million human leptospirosis cases , novel therapeutic approaches targeting queC could be significant.

What strategies can overcome poor expression or solubility of recombinant Leptospira queC?

When encountering expression or solubility issues:

• Expression optimization:

  • Test multiple E. coli strains (BL21, Arctic Express, Rosetta)

  • Vary induction conditions (temperature, IPTG concentration, duration)

  • Consider fusion partners (MBP, SUMO, TrxA) to enhance solubility

  • Co-express with molecular chaperones (GroEL/GroES)

• Solubility enhancement:

  • Optimize buffer conditions (pH, ionic strength, additives)

  • Include stabilizing agents (trehalose, glycerol, reducing agents)

  • Consider detergents for membrane-associated forms

  • Implement refolding protocols if inclusion bodies form

• Alternative approaches:

  • Cell-free expression systems

  • Homologous expression in Leptospira using IPTG-inducible systems

  • Expression of truncated constructs guided by bioinformatic analysis

How can the specificity and reproducibility of queC activity assays be improved?

To enhance assay performance:

• Substrate purity:

  • Synthesize or obtain high-purity 7-carboxy-7-deazaguanine

  • Validate substrate identity by NMR and mass spectrometry

  • Store under appropriate conditions to prevent degradation

• Assay optimization:

  • Implement internal standards for HPLC-MS based assays

  • Develop fluorescence-based assays for higher throughput

  • Control for non-enzymatic background reactions

  • Include positive controls with well-characterized queC enzymes

• Data analysis:

  • Apply appropriate kinetic models (Michaelis-Menten, substrate inhibition)

  • Use statistical methods to identify and manage outliers

  • Implement quality control metrics for assay validation

What are the critical parameters for successful crystallization of Leptospira queC?

For structural determination through crystallography:

• Protein preparation:

  • Achieve >95% purity through rigorous purification

  • Verify monodispersity by dynamic light scattering

  • Remove flexible regions identified through limited proteolysis

  • Stabilize protein through ligand binding (substrate analogs, ATP analogs)

• Crystallization screening:

  • Implement sparse matrix screens with varying precipitation agents

  • Test co-crystallization with substrates, products, and cofactors

  • Explore pH range 7.0-10.0 based on enzyme stability and activity profiles

  • Vary protein concentration (5-15 mg/mL) and temperature (4-20°C)

• Crystal optimization:

  • Fine-tune promising conditions through grid screens

  • Apply seeding techniques for improved crystal quality

  • Consider surface entropy reduction mutations

  • Explore crystallization additives to improve order

How does queC function differ between pathogenic and non-pathogenic Leptospira species?

Comparative analysis between pathogenic (e.g., L. interrogans) and non-pathogenic (e.g., saprophytic) Leptospira species may reveal important differences:

• Gene expression patterns:

  • Transcriptional regulation under various environmental conditions

  • Response to host-associated signals (temperature, osmolarity, serum)

• Enzyme characteristics:

  • Substrate affinity and catalytic efficiency

  • Temperature and pH optima reflecting ecological niches

  • Allosteric regulation and protein-protein interactions

• Physiological roles:

  • Impact on tRNA modification patterns

  • Contribution to stress response mechanisms

  • Influence on translation of specific virulence factors

This comparative approach can provide insights into Leptospira evolution and adaptation to different lifestyles, leveraging genome sequencing data from diverse Leptospira species .

What genomic context surrounds queC in Leptospira and how does this inform functional studies?

Analyzing the genomic neighborhood of queC provides crucial contextual information:

• Gene clustering:

  • Co-localization with other queuosine biosynthesis genes (queD, queE, queF)

  • Presence of tRNA modification enzymes

  • Proximity to metabolic pathway genes

• Regulatory elements:

  • Promoter analysis and transcription factor binding sites

  • Presence of riboswitches or attenuators

  • Small RNA interactions

• Evolutionary conservation:

  • Synteny analysis across Leptospira species

  • Horizontal gene transfer signatures

  • Selection pressure analysis

Whole genome sequencing of diverse Leptospira isolates, as prioritized by the leptospirosis research community , facilitates these comparative genomic approaches.

How can multi-omics approaches advance our understanding of queC function in Leptospira?

Integrating multiple omics technologies provides comprehensive insights:

• Genomics:

  • Whole genome sequencing to identify queC variants across strains

  • Comparative genomics to assess evolutionary conservation

• Transcriptomics:

  • RNA-seq to determine expression patterns under different conditions

  • Ribosome profiling to assess translational impacts

• Proteomics:

  • Global protein expression analysis in queC mutants

  • Protein-protein interaction studies to identify functional partners

  • Post-translational modification analysis

• Metabolomics:

  • Quantification of queuosine pathway intermediates

  • Global metabolic shifts in queC mutants

• Structural biology:

  • Protein structure determination

  • Molecular dynamics simulations of enzyme mechanism

This multi-omics strategy aligns with research priorities for understanding Leptospira biology and developing improved diagnostic and preventive measures for leptospirosis .