Recombinant Legionella pneumophila 2-nonaprenyl-3-methyl-6-methoxy-1,4-benzoquinol hydroxylase (coq7)

What is the functional role of coq7 in Legionella pneumophila biology?

Coq7 (2-nonaprenyl-3-methyl-6-methoxy-1,4-benzoquinol hydroxylase) functions as a critical enzyme in the ubiquinone biosynthesis pathway of Legionella pneumophila. Also known as 5-demethoxyubiquinone hydroxylase (DMQ hydroxylase), this enzyme catalyzes one of the final steps in the biosynthesis of ubiquinone, which is essential for electron transport chain function and bacterial energy metabolism . In the context of L. pneumophila pathogenesis, proper energy metabolism is crucial as the bacterium targets host cell mitochondria and modulates mitochondrial dynamics to impair respiration, creating favorable conditions for its intracellular replication .

What is the molecular structure and key characteristics of recombinant L. pneumophila coq7?

The recombinant L. pneumophila coq7 protein consists of 213 amino acids with the following sequence: MRTSSFLDRL IGEVDSALRT LVLPQKRITT RQSPAENLAD TVLSAQEKKH ISGLMRVNHA GEVCAQALYQ GQALTARLTH IKEQMASAAA EEVDHLAWCE ERLYELGSKP SLLNPIWYCG SVLLGALAGL AGDKISLGFV AETERQVTAH LQRHLHYLPE KDKKTIAILK RMQEDEEHHA HTAMEAGAVE LPYIIKQLMN AVSKLMTQSS YYI . The protein has been identified in the Legionella pneumophila strain Corby and is cataloged in the UniProt database under accession number A5I9H9 . The enzyme is classified with EC number 1.14.13.-, indicating it belongs to the oxidoreductase family acting on paired donors with incorporation of molecular oxygen .

What are the optimal conditions for expressing and purifying recombinant L. pneumophila coq7?

For optimal expression of recombinant L. pneumophila coq7, E. coli-based expression systems have proven effective . The protein can be expressed with various tags, with the specific tag type determined during the manufacturing process to optimize yield and solubility . For purification, standard affinity chromatography methods appropriate for the chosen tag system should be employed.

After purification, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard) and to aliquot the protein solution before storing at -20°C/-80°C . This prevents repeated freeze-thaw cycles that can compromise protein integrity. The purified protein typically demonstrates >85% purity as assessed by SDS-PAGE .

How can researchers verify the enzymatic activity of recombinant coq7 in vitro?

Researchers can verify the enzymatic activity of recombinant coq7 through several approaches:

• Spectrophotometric assays: Monitor the conversion of the substrate 2-nonaprenyl-3-methyl-6-methoxy-1,4-benzoquinol to its hydroxylated product by measuring changes in absorbance at specific wavelengths.

• HPLC analysis: Quantify substrate consumption and product formation using reverse-phase high-performance liquid chromatography.

• Coupled enzyme assays: Measure coq7 activity by linking its reaction to a secondary enzyme system that produces a detectable signal.

• Auto-thiophosphorylation assays: Similar to methods used with other Legionella kinases like LegK7, researchers can use thiophosphorylation techniques with recombinant coq7 to assess its activity . This approach may be adapted by using γ-[S]ATP and detecting thiophosphorylated proteins through alkylation and Western blotting.

• Pseudo-substrate phosphorylation: Test activity using a standard pseudo-substrate like myelin basic protein (Myelin A1), which has been successfully used with other Legionella enzymes .

What are the recommended storage conditions to maintain coq7 stability and activity?

To maintain the stability and activity of recombinant L. pneumophila coq7, the following storage conditions are recommended:

• Short-term storage: Working aliquots can be stored at 4°C for up to one week .

• Long-term storage:

  • For liquid preparations: Store at -20°C/-80°C with an expected shelf life of approximately 6 months .

  • For lyophilized preparations: Store at -20°C/-80°C with an expected shelf life of approximately 12 months .

• Handling precautions:

  • Briefly centrifuge vials before opening to bring contents to the bottom .

  • Avoid repeated freeze-thaw cycles as these significantly reduce enzyme activity .

  • When reconstituting lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL .

  • Add glycerol to a final concentration of 5-50% for cryoprotection before freezing .

How might coq7 interact with host mitochondrial function during L. pneumophila infection?

Given L. pneumophila's documented strategy of targeting host mitochondria, researchers should consider potential interactions between bacterial coq7 and host mitochondrial functions. L. pneumophila has been shown to change the shape of host mitochondria, impairing mitochondrial respiration and leading to metabolic changes that facilitate pathogen replication . While direct evidence of coq7's role in this process is limited in the provided search results, its function in ubiquinone biosynthesis suggests several hypothetical mechanisms:

• Competitive inhibition: Bacterial coq7 might compete with host mitochondrial coq7 (COQ7/CLK-1) for substrates, potentially disrupting host ubiquinone biosynthesis.

• Altered respiratory chain function: By contributing to changes in ubiquinone pools or ratios within infected cells, bacterial coq7 could indirectly affect mitochondrial respiratory chain complexes.

• Metabolic reprogramming: Changes in ubiquinone metabolism might contribute to the shift in host cell energy production observed during L. pneumophila infection, potentially favoring glycolysis over oxidative phosphorylation.

Further research using techniques such as mitochondrial respiration analysis, membrane potential measurements, and metabolomic profiling could help elucidate these potential interactions.

What approaches can be used to investigate potential cross-talk between coq7 and L. pneumophila effector proteins?

Investigating potential cross-talk between coq7 and L. pneumophila effector proteins requires sophisticated experimental approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation assays to identify physical interactions

  • Yeast two-hybrid or bacterial two-hybrid screening

  • Proximity labeling techniques (BioID, APEX) in infection models

  • Fluorescence resonance energy transfer (FRET) microscopy

• Functional genomics approaches:

  • Construction of bacterial mutants with combinations of effector gene deletions including coq7

  • Complementation studies with wild-type and mutant versions of coq7

  • RNAseq to identify transcriptional changes when coq7 is expressed alongside various effectors

• Metabolic analysis:

  • Metabolomics to track changes in key metabolites when coq7 and effector proteins are present

  • Stable isotope labeling to follow metabolic flux through pathways potentially affected by coq7 and effectors

• Structural biology:

  • Crystallography or cryo-EM studies of coq7 alone and in complex with potential effector proteins

  • Molecular docking simulations to predict interactions

Since L. pneumophila effectors like LegK7 can hijack host signaling pathways , researchers should examine whether coq7 activity influences or is influenced by such effector-mediated signaling events.

What are the challenges in distinguishing between host and bacterial coq7 activities during infection studies?

Distinguishing between host and bacterial coq7 activities during infection presents several significant challenges:

• Sequence and functional similarity: Human COQ7 and bacterial coq7 catalyze similar reactions, making biochemical differentiation challenging.

• Sample preparation issues:

  • Subcellular fractionation may not cleanly separate bacterial and host components

  • Protein extraction methods may favor one source over the other

• Antibody cross-reactivity: Antibodies raised against either human or bacterial coq7 may cross-react due to conserved epitopes.

• Methodological approaches to overcome these challenges:

  • Use of epitope-tagged bacterial coq7 in recombinant strains

  • CRISPR-Cas9 editing of host cells to tag endogenous COQ7

  • Selective inhibition using species-specific inhibitors (when available)

  • Mass spectrometry-based approaches to distinguish protein variants based on unique peptide signatures

  • Species-specific PCR or RT-qPCR to quantify gene expression from each source

• Control experiments:

  • Include uninfected controls and controls with bacterial mutants lacking coq7

  • Use purified recombinant proteins from both species as standards for activity assays

How does L. pneumophila coq7 compare structurally and functionally to its human homolog?

The L. pneumophila coq7 (2-nonaprenyl-3-methyl-6-methoxy-1,4-benzoquinol hydroxylase) shares fundamental catalytic properties with its human homolog COQ7/CLK-1, yet exhibits distinct structural features. Both enzymes catalyze a critical hydroxylation step in ubiquinone biosynthesis, but differences in substrate specificity, regulation, and inhibitor sensitivity exist.

From a structural perspective, L. pneumophila coq7 consists of 213 amino acids , while the human homolog is typically larger with additional regulatory domains. The bacterial enzyme lacks some of the sophisticated regulatory elements found in the eukaryotic version, reflecting the less complex regulatory requirements of bacterial metabolism compared to compartmentalized eukaryotic systems.

Key differences that researchers should consider when working with these homologs include:

• Substrate preferences: The bacterial enzyme typically works with shorter prenyl chains compared to the human enzyme.

• Cofactor requirements: Both require iron for catalytic activity, but may differ in their affinity for other cofactors.

• Inhibitor sensitivity: The structural differences likely translate to differential sensitivity to small molecule inhibitors, potentially providing opportunities for selective targeting in therapeutic development.

• Regulatory mechanisms: The human enzyme is subject to complex post-translational modifications and regulatory interactions that are likely absent in the bacterial version.

What is the relationship between coq7 and other proteins in the L. pneumophila ubiquinone biosynthesis pathway?

The L. pneumophila ubiquinone biosynthesis pathway involves a coordinated series of enzymatic reactions with coq7 playing a crucial role in the later stages. Key relationships include:

• Pathway position: Coq7 catalyzes the penultimate step in ubiquinone biosynthesis, hydroxylating 5-demethoxyubiquinone (DMQ) to form 5-hydroxyubiquinone .

• Substrate dependency: Coq7 requires the products of upstream enzymes in the pathway, including those responsible for prenylation and ring modification steps.

• Product utilization: The hydroxylated product of the coq7 reaction serves as the substrate for the final methylation step, catalyzed by coq3.

• Potential protein-protein interactions: In eukaryotes, Coq proteins form a complex (the "CoQ-synthome"); whether similar complexes exist in L. pneumophila is not well established but represents an important research question.

• Regulatory relationships: Expression of coq7 likely coordinates with other enzymes in the pathway, potentially through shared transcriptional regulators.

Understanding these relationships is crucial for comprehending how L. pneumophila maintains adequate ubiquinone levels for energy metabolism during infection and intracellular growth.

What role might coq7 play in L. pneumophila's adaptation to different intracellular environments?

L. pneumophila must adapt to diverse intracellular environments during its infectious cycle, and coq7 may play a significant role in this adaptation process. The bacterium's ability to modulate host cell metabolism by targeting mitochondria suggests a sophisticated adaptation mechanism that could involve ubiquinone metabolism.

Potential roles for coq7 in environmental adaptation include:

• Metabolic flexibility: Varying ubiquinone levels might allow L. pneumophila to adjust its energy metabolism in response to changing nutrient availability within different host cell types or intracellular compartments.

• Oxidative stress response: As ubiquinone serves as an antioxidant, coq7 activity might be modulated to adjust bacterial defenses against varying levels of oxidative stress encountered in different host environments.

• Membrane composition adaptation: Ubiquinone contributes to membrane properties, and changes in its synthesis through coq7 activity could help the bacterium adapt its membrane characteristics to different intracellular conditions.

• Integration with sensing systems: Coq7 activity might be regulated in response to environmental sensing systems that detect conditions within the host cell, allowing coordinated metabolic adaptation.

Researchers investigating these possibilities might employ techniques like selective mutants with conditionally regulated coq7 expression, metabolomic profiling across infection stages, and comparative studies across different host cell types.

How can structural biology approaches enhance our understanding of L. pneumophila coq7?

Structural biology approaches offer powerful means to deepen our understanding of L. pneumophila coq7:

The full-length protein sequence information provides a starting point for structural studies, and determination of high-resolution structures would significantly advance our understanding of this enzyme's function in L. pneumophila biology.

What technological advancements might improve detection and quantification of coq7 activity in complex biological samples?

Several emerging technologies and methodological advances could enhance detection and quantification of coq7 activity in complex biological samples:

• Advanced mass spectrometry approaches:

  • Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for specific detection of coq7 peptides

  • Activity-based protein profiling coupled with mass spectrometry

  • Isotope-coded activity-based probes to distinguish between active and inactive enzyme populations

• Biosensor development:

  • FRET-based sensors that respond to coq7 activity

  • Genetically encoded biosensors that can report on coq7 activity in living cells

  • Nanobody-based detection systems with enhanced specificity

• Single-cell technologies:

  • Single-cell metabolomics to detect ubiquinone intermediates in individual infected cells

  • Multiplexed imaging mass cytometry to simultaneously visualize multiple components of the ubiquinone pathway

  • Single-cell proteomics to quantify coq7 in individual bacteria within host cells

• Digital microfluidics:

  • Droplet-based assays for high-throughput screening of coq7 activity

  • Microfluidic devices for monitoring enzyme kinetics in real-time

• Computational approaches:

  • Machine learning algorithms to identify coq7 activity signatures in complex datasets

  • Integrative multi-omics approaches to correlate coq7 activity with other cellular processes

These technological advancements would enable researchers to better understand the spatial and temporal dynamics of coq7 activity during L. pneumophila infection, providing new insights into the enzyme's role in pathogenesis.