Recombinant Desulfovibrio vulgaris 7-carboxy-7-deazaguanine synthase (queE)

What is the function of 7-carboxy-7-deazaguanine synthase (queE) in bacterial metabolism?

7-Carboxy-7-deazaguanine synthase (queE) catalyzes the radical-mediated ring contraction of 6-carboxy-5,6,7,8-tetrahydropterin, forming the characteristic pyrrolopyrimidine core found in all 7-deazaguanine natural products . This reaction represents a critical step in the biosynthesis of queuosine, a modified nucleoside present in the wobble position of certain tRNAs in bacteria. As a member of the S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily, queE harnesses the reactivity of radical intermediates to perform this challenging chemical transformation .

Recent research has revealed that queE possesses a secondary "moonlighting" function beyond its primary biosynthetic role. When expressed at high levels, queE can co-localize with the cell division protein FtsZ, inhibiting cell division and resulting in filamentous growth . This dual functionality makes queE particularly interesting from both biochemical and cellular biology perspectives.

How does the structure of queE relate to its catalytic mechanism?

QueE belongs to the AdoMet radical enzyme superfamily, which is characterized by a canonical binding motif (CX3CX2C) . This motif coordinates an iron-sulfur cluster that is essential for the enzyme's radical-based chemistry. The enzyme uses this [4Fe-4S] cluster to cleave AdoMet, generating a reactive 5'-deoxyadenosyl radical that initiates the ring contraction reaction.

Structural studies of queE homologs have revealed variations in the architecture that may influence interactions with the biological reductant, flavodoxin . These structural variations are likely important for electron transfer during catalysis, as the iron-sulfur cluster must be reduced to the active state before it can cleave AdoMet.

The catalytic core of queE contains binding sites for AdoMet, the substrate, and the iron-sulfur cluster in a configuration that facilitates radical transfer between these components during the reaction cycle.

How do researchers distinguish between the primary and secondary functions of queE?

Distinguishing between queE's biosynthetic role and its cell division regulatory function requires careful experimental design:

• Concentration dependence: The secondary function as a cell division regulator manifests at high expression levels, whereas the primary enzymatic function occurs at physiological concentrations .

• Localization patterns: When functioning as a division regulator, queE co-localizes with FtsZ at the septal site, whereas its biosynthetic role involves a more diffuse cytoplasmic distribution .

• Mutational analysis: Alanine scanning mutagenesis has identified specific residues that contribute differentially to each function, establishing queE as a true moonlighting protein . Some mutations affect only one function while preserving the other.

• Comparative biology: QueE orthologs from enterobacteria (Salmonella typhimurium and Klebsiella pneumoniae) share the cell division regulatory capability, while more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this secondary function . This evolutionary pattern helps distinguish between conserved primary and more recently evolved secondary functions.

What are the optimal expression systems for recombinant Desulfovibrio vulgaris queE?

Based on experiences with other recombinant proteins from Desulfovibrio vulgaris, particularly iron-sulfur proteins like rubrerythrin, the following expression strategy is recommended:

• Expression host: Escherichia coli BL21(DE3) or similar strains offer good expression levels for Desulfovibrio proteins . For queE specifically, strains optimized for iron-sulfur protein expression (containing the isc or suf operons) may improve yield of properly folded protein.

• Vector selection: pET-based vectors with T7 promoter systems provide strong, inducible expression that can be modulated by varying IPTG concentration .

• Growth conditions: Rich media supplemented with iron (e.g., ferric ammonium citrate) and cysteine can enhance iron-sulfur cluster incorporation. Growing cells at reduced temperatures (16-25°C) after induction can improve protein solubility.

• Induction parameters: Low concentrations of IPTG (0.1-0.5 mM) and extended expression times (12-16 hours) at lower temperatures often yield better results for complex metalloproteins .

• Cell lysis considerations: As observed with rubrerythrin, overexpressed iron-sulfur proteins often form inclusion bodies deficient in iron . Gentle lysis methods and/or in vitro iron incorporation protocols may be necessary.

What strategies are effective for reconstituting the iron-sulfur cluster in recombinant queE?

When expressed in E. coli, iron-sulfur proteins like queE often require in vitro reconstitution of their metal centers. Based on protocols developed for similar proteins, the following approach is recommended:

• Solubilization of inclusion bodies: If the protein is insoluble, dissolve using 3-6 M guanidinium chloride or 8 M urea under anaerobic conditions .

• Protein refolding: Gradually dilute the denaturant through dialysis or by sequential dilution steps while maintaining strictly anaerobic conditions .

• Iron incorporation: Add Fe(II) (typically as ferrous ammonium sulfate) anaerobically at a molar ratio of 6-10 iron atoms per protein monomer . This excess accounts for incomplete incorporation.

• Sulfide addition: Introduce sodium sulfide in equimolar amounts to iron to facilitate iron-sulfur cluster formation.

• Incubation conditions: Maintain the reconstitution mixture at 4°C for 12-24 hours under anaerobic conditions with gentle stirring.

• Removal of excess reagents: Dialyze or use size-exclusion chromatography to remove unincorporated iron and sulfide.

• Verification: Confirm successful reconstitution through UV-visible spectroscopy, which should show characteristic absorption features of [4Fe-4S] clusters (broad absorption around 390-420 nm) .

How can researchers verify the integrity of the iron-sulfur cluster in purified queE?

Multiple complementary spectroscopic techniques are essential for comprehensive characterization of the iron-sulfur cluster in queE:

• UV-visible absorption spectroscopy provides initial confirmation of cluster incorporation, with [4Fe-4S] clusters typically showing broad absorption bands around 390-420 nm .

• Electron Paramagnetic Resonance (EPR) spectroscopy is crucial for characterizing the redox state of the cluster. The [4Fe-4S]+ state exhibits characteristic g-values (typically around g = 2.03, 1.93, and 1.87), while the [4Fe-4S]2+ state is EPR-silent .

• Mössbauer spectroscopy offers detailed information about iron oxidation states and coordination environments. The isomer shift (δ) and quadrupole splitting (ΔEQ) parameters can distinguish different types of iron centers .

• Iron quantification using colorimetric assays (e.g., ferene method) or inductively coupled plasma mass spectrometry (ICP-MS) can determine the iron:protein ratio, which should be approximately 4:1 for a [4Fe-4S] cluster per monomer.

• Activity assays measuring AdoMet cleavage and/or substrate conversion provide functional verification of the reconstituted enzyme.

What are the best methods for studying the interaction between queE and its biological reductant?

The interaction between queE and its biological reductant (likely flavodoxin based on studies of homologous enzymes) is critical for understanding the complete catalytic cycle:

• Co-crystallization and structural analysis: Attempts to co-crystallize queE with flavodoxin can provide atomic-level details of the interaction interface . Comparative analysis of multiple queE homologs suggests structural variations that may influence flavodoxin binding .

• Surface plasmon resonance (SPR): This technique enables real-time monitoring of protein-protein interactions and determination of binding kinetics and affinity constants under various conditions.

• Isothermal titration calorimetry (ITC): Provides thermodynamic parameters (ΔH, ΔS, ΔG) for the interaction, offering insights into the driving forces behind complex formation.

• Site-directed mutagenesis: Based on structural predictions or homology models, mutation of potential interface residues can identify key determinants of the interaction . Both kinetic and binding assays with mutant proteins can map the interaction surface.

• Redox potential measurements: Protein film voltammetry or spectroelectrochemical techniques can determine the redox potentials of both queE and flavodoxin, providing insights into the thermodynamic favorability of electron transfer.

• Electron transfer kinetics: Stopped-flow spectroscopy can monitor the reduction of the iron-sulfur cluster by flavodoxin in real-time, elucidating the kinetics of this critical step.

How should researchers design experiments to investigate the moonlighting function of queE?

Based on successful approaches with E. coli queE, the following experimental design principles are recommended:

• Expression level control: Use precisely tunable expression systems to establish the concentration-dependence of queE's effects on cell division . Quantitative Western blotting or fluorescence measurements can correlate protein levels with phenotypic outcomes.

• Fluorescence microscopy: Utilize fluorescently tagged queE constructs to visualize subcellular localization and potential co-localization with FtsZ or other division proteins . Time-lapse microscopy can reveal the dynamics of this interaction during the cell cycle.

• Structure-function analysis: Employ a systematic alanine scanning mutagenesis approach to identify residues specifically involved in cell division regulation versus catalytic activity . This allows separation of the two functions for independent study.

• Domain mapping: Compare the sequences of queE orthologs with and without the cell division regulatory capability to identify regions specific to this function . Construction of chimeric proteins can test hypotheses about these domains.

• Protein-protein interaction studies: Use bacterial two-hybrid systems, co-immunoprecipitation, or proximity labeling approaches to identify the full complement of proteins interacting with queE in its different functional modes.

• In vitro reconstitution: Attempt to reconstitute the effect on FtsZ polymerization or function using purified components to establish a direct mechanistic link.

What crystallization strategies are most promising for obtaining structural information about queE?

Based on experiences with other Desulfovibrio vulgaris metalloproteins like rubrerythrin, the following crystallization strategies may prove successful:

How should researchers interpret spectroscopic changes during queE catalysis?

The interpretation of spectroscopic data during queE catalysis requires understanding the signatures of different states in the catalytic cycle:

• EPR spectral changes: The appearance or disappearance of specific g-values can indicate changes in the redox state of the [4Fe-4S] cluster. The reduced [4Fe-4S]+ state typically shows a characteristic rhombic signal (g ≈ 2.03, 1.93, 1.87) that disappears upon AdoMet binding as the electron is transferred for homolytic cleavage.

• UV-visible spectral shifts: Changes in the broad absorption features of the iron-sulfur cluster (390-420 nm) upon substrate or AdoMet binding can provide insights into alterations in the cluster environment during catalysis.

• Mössbauer parameter analysis: Changes in isomer shift (δ) and quadrupole splitting (ΔEQ) values reflect alterations in iron oxidation states and coordination environments throughout the catalytic cycle . For instance, the partial reduction of diiron sites in Desulfovibrio vulgaris rubrerythrin resulted in identifiable changes in these parameters .

• Time-resolved measurements: Using rapid freeze-quench techniques coupled with EPR or Mössbauer spectroscopy can capture transient intermediates in the catalytic cycle, revealing the sequence of electron transfer events.

• Comparative analysis: Comparing spectral changes with those observed in well-characterized AdoMet radical enzymes can help identify common features versus unique aspects of queE catalysis.

What computational approaches complement experimental studies of queE?

Computational methods provide valuable insights that enhance experimental investigations of queE:

• Homology modeling: When crystal structures are unavailable, models based on related AdoMet radical enzymes can provide preliminary structural insights. Multiple sequence alignments of queE orthologs can identify conserved features versus variable regions that may correlate with functional differences .

• Molecular dynamics simulations: These can reveal protein flexibility, conformational changes during catalysis, and potential interaction surfaces for flavodoxin binding or FtsZ interaction. Simulations can also model the effects of mutations identified in experimental studies.

• Quantum mechanical calculations: Essential for modeling the radical-based reaction mechanism, particularly the challenging ring contraction step catalyzed by queE. QM/MM approaches can incorporate the protein environment while focusing computational resources on the active site.

• Docking studies: Predict binding modes of substrates, AdoMet, and protein partners like flavodoxin or FtsZ. These studies can guide the design of experiments to test specific interaction hypotheses.

• Network analysis: Identify potential allosteric pathways connecting the catalytic site with regions involved in the secondary cell division regulatory function, helping to explain how mutations can selectively affect one function over the other.

• Phylogenetic analysis: Trace the evolutionary history of queE across different bacterial lineages, potentially revealing when and how the secondary function emerged in certain lineages but not others .

How can new methodologies advance our understanding of queE's dual functionality?

Several emerging technologies hold promise for deeper insights into queE's structure-function relationships:

• Cryo-electron microscopy (cryo-EM): As resolution capabilities improve, cryo-EM could reveal the structural basis of queE's interaction with the cell division machinery, which may be difficult to capture using crystallography due to the size and complexity of these assemblies.

• Single-molecule techniques: Single-molecule FRET or force spectroscopy could detect conformational changes in queE associated with switching between its dual functions, providing dynamic information not accessible through bulk methods.

• Native mass spectrometry: Could characterize the stoichiometry and stability of queE complexes with various partners under near-physiological conditions, helping to elucidate its protein interaction network.

• CRISPR-based approaches: Precise genome editing to modify endogenous queE levels or introduce specific mutations could reveal how its dual functionality operates in the native cellular context.

• Proteomics and interactomics: Comprehensive analysis of queE's protein interaction network under different conditions could identify additional partners beyond FtsZ that contribute to its cellular functions.

• Time-resolved structural methods: X-ray free-electron lasers or time-resolved crystallography could potentially capture structural snapshots during catalysis, revealing conformational changes associated with the radical reaction mechanism.

What are the implications of queE's dual functionality for understanding bacterial stress responses?

The discovery that queE functions as both a biosynthetic enzyme and a cell division regulator has significant implications for bacterial physiology:

• Integrated stress response: QueE's upregulation during exposure to certain stressors (such as cationic antimicrobial peptides) suggests a mechanism by which bacteria coordinate RNA modification and cell division in response to environmental challenges .

• Evolutionary adaptation: The presence of this secondary function in enterobacterial queE orthologs but not in more distant bacteria suggests relatively recent evolutionary acquisition of this capability, potentially as an adaptation to specific ecological niches .

• Moonlighting protein network: QueE may be part of a broader network of proteins with dual functionalities that collectively regulate bacterial responses to changing environments. Identifying other components of this network could reveal new regulatory paradigms.

• Queuosine modification significance: The link between queuosine biosynthesis and cell division regulation suggests potentially unexplored roles for this tRNA modification in coordinating translation with cell cycle progression.

• Stress-induced filamentation: The mechanism by which queE induces filamentous growth by interacting with FtsZ represents a novel pathway for stress-induced filamentation, distinct from previously characterized SOS-dependent mechanisms .

Understanding these implications could lead to new strategies for manipulating bacterial growth and adaptation, with potential applications in biotechnology and antimicrobial development.