Recombinant Chromobacterium violaceum Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the basic function of UbiB in Chromobacterium violaceum?

UbiB in C. violaceum functions as a crucial enzyme in the ubiquinone (coenzyme Q) biosynthesis pathway, specifically involved in the first monooxygenase step. This protein belongs to a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member . UbiB catalyzes the conversion of the biosynthetic intermediate octaprenylphenol to the subsequent intermediates in the pathway, making it essential for respiratory electron transport chain function. Disruption of UbiB in related bacteria results in the accumulation of octaprenylphenol and absence of coenzyme Q, demonstrating its critical role in this pathway .

To study this function experimentally, researchers typically use gene knockout studies followed by chromatographic analysis of accumulated intermediates. Comparative analysis with E. coli UbiB (formerly known as yigR) provides valuable insights, as demonstrated in complementation studies where the genes show functional conservation across bacterial species.

How is the ubiB gene organized within the C. violaceum genome?

Based on data from related bacterial systems, the ubiB gene in C. violaceum is likely organized within an operon structure. In E. coli, ubiB is the third gene in an operon containing ubiE, yigP, and ubiB, with ubiE encoding a C-methyltransferase required for both coenzyme Q and menaquinone synthesis . This organization suggests coordinated expression of multiple ubiquinone biosynthesis genes.

Experimental verification of this organization in C. violaceum would require techniques such as:

• Reverse transcription PCR to identify co-transcribed genes

• Promoter mapping using 5' RACE (Rapid Amplification of cDNA Ends)

• Northern blot analysis to identify transcriptional units

• Genome walking to confirm the presence of adjacent genes in the operon

Understanding this organization is crucial for designing expression constructs that maintain natural regulation when producing recombinant UbiB.

What expression systems are most suitable for producing recombinant C. violaceum UbiB?

For successful recombinant expression of C. violaceum UbiB, several expression systems have proven effective for similar proteins:

When designing expression constructs, researchers should consider using vectors with N-terminal His6-tags for purification, as C-terminal tags may interfere with protein function based on predicted structural properties of UbiB. Temperature optimization is critical, with most successful expressions occurring at lower temperatures (16-25°C) to ensure proper folding.

How does the predicted protein kinase activity of UbiB contribute to ubiquinone biosynthesis?

UbiB belongs to a family of predicted protein kinases that includes the prototypic Saccharomyces cerevisiae ABC1 gene . While traditional protein kinase activity (phosphorylation of protein substrates) has not been definitively demonstrated for UbiB, research suggests several possible mechanisms:

• UbiB may phosphorylate other ubiquinone biosynthetic enzymes, regulating their activity through post-translational modification

• UbiB might phosphorylate biosynthetic intermediates in the ubiquinone pathway rather than proteins

• The kinase-like domain may be involved in ATP binding rather than phosphotransfer, providing energy for monooxygenase activity

Experimental approaches to investigate this function include:

• In vitro kinase assays with purified recombinant UbiB and potential substrates

• Mutagenesis of predicted catalytic residues in the kinase domain followed by functional complementation assays

• Metabolomic analysis to identify phosphorylated intermediates in the ubiquinone pathway

• Structural studies using X-ray crystallography or cryo-EM to visualize substrate binding sites

The precise biochemical mechanism of UbiB remains one of the most significant unanswered questions in ubiquinone biosynthesis research.

What structural features distinguish C. violaceum UbiB from UbiB proteins in other bacterial species?

Comparative structural analysis of UbiB proteins reveals both conserved and species-specific features:

Structural predictions suggest that the kinase domain is highly conserved across species, while C-terminal regions show greater variability, potentially reflecting adaptation to different substrates or protein-protein interactions specific to each organism's ubiquinone biosynthetic pathway.

Advanced structural studies using hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with molecular dynamics simulations would be valuable for identifying flexible regions and substrate-binding sites that might explain functional differences between UbiB homologs.

How does the redox environment affect UbiB activity and stability?

UbiB functions in ubiquinone biosynthesis, a pathway intrinsically linked to cellular redox processes. Research indicates that the redox environment significantly impacts UbiB stability and function:

• Presence of reducing agents (DTT, β-mercaptoethanol) at 1-5 mM in purification buffers improves protein stability

• UbiB activity is optimal under microaerobic conditions, suggesting oxygen sensitivity

• Cysteine residues in UbiB are likely involved in maintaining proper protein conformation

Experimental approaches to study redox sensitivity include:

• Site-directed mutagenesis of conserved cysteine residues

• Activity assays under varying redox conditions

• Differential scanning fluorimetry to assess thermal stability in different redox environments

• Mass spectrometry to identify post-translational modifications related to oxidative stress

Researchers working with recombinant UbiB should carefully control the redox environment during purification and storage to maintain functional protein.

What are the most effective methods for purification of recombinant C. violaceum UbiB?

Purification of recombinant C. violaceum UbiB requires specialized approaches due to its predicted membrane association and potential instability:

Throughout purification, maintaining a temperature of 4°C and including protease inhibitors is essential. The addition of mild detergents (0.01-0.05% DDM or CHAPS) can improve protein solubility without disrupting function. Researchers should avoid freeze-thaw cycles, as they significantly reduce UbiB activity.

What assays can be used to measure UbiB enzymatic activity in vitro?

Several complementary approaches can be used to assess UbiB activity:

• Substrate conversion assay: Monitoring the conversion of octaprenylphenol to the subsequent intermediate using HPLC or LC-MS. This direct approach requires synthesized substrate and analytical standards.

• ATP consumption assay: If UbiB functions as a kinase, ATP consumption can be measured using coupled enzyme assays (e.g., pyruvate kinase/lactate dehydrogenase system) or radioactive [γ-32P]ATP.

• Oxygen consumption assay: Using an oxygen electrode to measure consumption during the monooxygenase reaction.

• Complementation assays: Functional complementation of E. coli ubiB mutants, measuring restoration of ubiquinone biosynthesis.

For a comprehensive assessment of UbiB activity, researchers should combine multiple assay types and validate using site-directed mutagenesis of predicted catalytic residues.

How can researchers implement CRISPR-Cas9 techniques to study UbiB function in C. violaceum?

CRISPR-Cas9 genome editing provides powerful tools for studying UbiB function in C. violaceum, allowing precise genetic modifications:

• Design of guide RNAs: Target sequences should be 20 nucleotides followed by NGG PAM sequence, with high specificity scores to avoid off-target effects. Multiple bioinformatic tools can identify optimal sgRNA sequences in the C. violaceum genome.

• Delivery methods:

  • Electroporation of ribonucleoprotein complexes (Cas9 protein + sgRNA)

  • Conjugation using broad-host-range plasmids containing Cas9 and sgRNA expression cassettes

• Editing strategies:

  • Gene disruption: Introduction of frameshift mutations or early stop codons

  • Point mutations: Homology-directed repair using repair templates to introduce specific amino acid changes

  • Domain swapping: Replacing domains with homologous regions from other species

• Phenotypic analysis:

  • Growth curves under various conditions

  • Metabolomic analysis to identify accumulated intermediates

  • Complementation with wild-type or mutant alleles

When implementing CRISPR in C. violaceum, researchers should consider using inducible promoters for Cas9 expression to minimize toxicity and optimize transformation conditions for this particular species.

How does C. violaceum UbiB compare functionally to homologs in model organisms?

Comparative functional analysis reveals both conservation and divergence among UbiB homologs:

Experimental approaches to study functional conservation include heterologous expression of C. violaceum UbiB in deficient strains of other organisms, followed by metabolomic analysis to assess restoration of ubiquinone biosynthesis. Cross-species complementation studies provide valuable insights into which domains and residues are essential for function across evolutionary distance.

How does the genomic context of ubiB differ between C. violaceum and related bacteria?

The genomic context of ubiB varies across bacterial species, with important implications for regulation and function:

The evolutionary conservation of the ubiE-yigP-ubiB operon structure between E. coli and potentially C. violaceum suggests functional coupling of these genes in ubiquinone biosynthesis. Researchers interested in expression regulation should examine the upstream regions for binding sites of regulatory proteins known in C. violaceum, particularly those involved in quorum sensing like the CviI/R system .

What insights about UbiB can be derived from studying the pathogenicity of C. violaceum?

C. violaceum is an opportunistic pathogen that can cause severe infections in humans, with a high mortality rate . The relationship between UbiB function and pathogenicity provides several research avenues:

• Metabolic adaptation during infection: Ubiquinone biosynthesis may be critical for adaptation to host environments where oxygen levels fluctuate.

• Stress response: UbiB function may contribute to oxidative stress resistance during host immune response.

• Virulence factor regulation: The redox state influenced by ubiquinone may affect expression of virulence factors, including the two type III secretion systems present in C. violaceum .

• Potential drug target: The essential nature of UbiB for bacterial respiration makes it a potential target for antimicrobial development.

Experimental approaches could include:

• Infection models using wildtype and UbiB-deficient C. violaceum

• Transcriptomic comparison under host-like conditions

• Assessment of virulence factor production in UbiB mutants

Understanding the link between primary metabolism (ubiquinone biosynthesis) and pathogenicity could reveal new therapeutic strategies against C. violaceum infections, which though rare, have high mortality rates .

How does UbiB interact with other proteins in the ubiquinone biosynthetic pathway?

UbiB likely functions as part of a multi-protein complex in the ubiquinone biosynthetic pathway:

• Predicted protein-protein interactions:

  • Direct interaction with UbiA (prenyl transferase)

  • Potential complex formation with UbiX/UbiD (decarboxylases)

  • Possible interaction with UbiE (methyltransferase) supported by operon organization

• Experimental approaches to study interactions:

  • Bacterial two-hybrid screening

  • Pull-down assays with tagged UbiB as bait

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Blue native PAGE to identify native complexes

• Functional significance of interactions:

  • Substrate channeling between enzymes

  • Regulation of enzymatic activities

  • Localization of pathway components to specific membrane domains

Researchers should consider that membrane association of many ubiquinone biosynthesis enzymes complicates interaction studies, requiring specialized approaches like membrane-based two-hybrid systems or in situ proximity labeling techniques.

What role does UbiB play in cellular response to oxidative stress?

UbiB's function in ubiquinone biosynthesis connects it to cellular redox homeostasis and oxidative stress response:

The relationship between UbiB and oxidative stress involves multiple mechanisms:

• Ubiquinone serves as an electron carrier and antioxidant

• UbiB activity may be directly regulated by redox-sensitive residues

• Ubiquinone levels affect the expression of stress response genes

Experimental approaches should include transcriptomics and proteomics of wildtype vs. UbiB mutants under oxidative stress conditions, combined with detailed phenotypic characterization and direct measurement of reactive oxygen species.

How can structural biology techniques be applied to understand UbiB catalytic mechanism?

Advanced structural biology approaches provide critical insights into UbiB function:

• X-ray crystallography challenges and solutions:

  • Challenge: Membrane association complicates crystallization

  • Solution: Construct design removing membrane-binding regions while preserving catalytic domains

  • Alternative: Lipidic cubic phase crystallization that accommodates membrane proteins

• Cryo-electron microscopy (cryo-EM):

  • Single-particle analysis for high-resolution structure

  • Visualization of UbiB in complex with other pathway enzymes

  • Sample preparation using nanodiscs to maintain native-like membrane environment

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Solution NMR for dynamics studies of soluble domains

  • Solid-state NMR for membrane-associated regions

  • NMR metabolomics to track conversion of isotope-labeled substrates

• Computational approaches:

  • Molecular dynamics simulations to model substrate binding and catalysis

  • Quantum mechanics/molecular mechanics (QM/MM) to model electron transfer

  • AlphaFold2 or RoseTTAFold predictions as starting models for experimental validation

Integration of multiple structural approaches, combined with functional assays on structure-based mutants, provides the most comprehensive understanding of UbiB's catalytic mechanism.