Recombinant Escherichia coli O7:K1 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is UbiB and what is its primary function in E. coli?

UbiB (formerly annotated as yigR) is a protein required for the first monooxygenase step in coenzyme Q (ubiquinone, CoQ) biosynthesis in Escherichia coli. It was initially identified through genetic studies of CoQ-deficient mutants. The identification of UbiB as the E. coli homologue of the aarF gene from Providencia stuartii was a significant step in understanding its role .

Both P. stuartii aarF and E. coli ubiB disruption mutant strains lack CoQ and accumulate a biosynthetic intermediate called octaprenylphenol, indicating the critical role of UbiB in hydroxylation reactions during CoQ biosynthesis . UbiB belongs to a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member, suggesting potential regulatory functions beyond direct catalysis .

While early research proposed that UbiB was responsible for C5-hydroxylation in the CoQ biosynthetic pathway, more recent studies indicate that this specific hydroxylation is actually performed by UbiI, highlighting the evolving understanding of UbiB's precise function .

How is the ubiB gene organized in the E. coli genome?

The ubiB gene in E. coli is part of an operon structure that includes multiple genes involved in ubiquinone biosynthesis. Specifically, ubiB is located downstream of ubiE and yigP in an operon containing ubiE, yigP, and ubiB . This genomic organization is functionally significant as demonstrated by studies of mutations affecting this region.

Analysis of the E. coli strain AN59, which contains the ubiB409 mutant allele, revealed that the strain's inability to synthesize CoQ was not due to mutations within the ubiB gene itself, but rather to an IS1 element insertion at position +516 of the ubiE gene . The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene .

Prior research incorrectly annotated yigQ and yigR as separate genes, but sequence analysis indicates that they actually correspond to one contiguous coding region, now referred to as ubiB . This clarification of the gene structure has been crucial for understanding the function and regulation of UbiB.

What methods are effective for recombinant UbiB production and characterization?

Recombinant UbiB protein can be effectively produced using baculovirus expression systems, which allow for proper folding and potential post-translational modifications . When working with recombinant UbiB, researchers should consider the following methodological approaches:

• Expression System Selection: Baculovirus systems yield high purity (>85% as determined by SDS-PAGE) recombinant protein suitable for functional studies .

• Protein Storage: The shelf life of liquid UbiB preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms can be stable for up to 12 months at the same temperatures .

• Reconstitution Protocol: Prior to use, centrifuge vials briefly and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% and aliquoting for long-term storage at -20°C/-80°C is recommended to maintain stability .

• Handling Precautions: Repeated freezing and thawing should be avoided. Working aliquots can be stored at 4°C for up to one week .

• Functional Assays: Due to UbiB's role in ubiquinone biosynthesis, functional characterization typically involves complementation studies in UbiB-deficient strains and analysis of ubiquinone levels via HPLC or LC-MS methods.

How does UbiB differ from other proteins in the CoQ biosynthesis pathway?

UbiB has unique characteristics that distinguish it from other Ubi proteins involved in the CoQ biosynthesis pathway:

• Sequence Classification: Unlike UbiH and UbiF, which are identified as class A flavoprotein monooxygenases based on sequence analysis, UbiB does not contain motifs characteristic of hydroxylases . Instead, it displays conserved motifs found in a superfamily of ancestral kinases .

• Functional Role: While UbiH and UbiF catalyze specific hydroxylation reactions (C1- and C6-hydroxylation, respectively), UbiB's precise catalytic function remains less clear . Early studies suggested UbiB's involvement in C5-hydroxylation based on the accumulation of octaprenylphenol (OPP) in ubiB⁻ strains, but this association has been questioned as strains deficient in the methyltransferase UbiG also accumulate OPP .

• Genetic Organization: UbiB is part of the ubiE-yigP-ubiB operon, indicating coordinated expression with these related genes . In contrast, other Ubi genes like ubiH and ubiF are located elsewhere in the genome.

• Evolutionary Conservation: UbiB belongs to a protein family that includes the Saccharomyces cerevisiae ABC1 gene, suggesting a conserved function across diverse organisms . This conservation pattern differs from that of other Ubi proteins.

What experimental approaches can resolve contradictions about UbiB's role in C5-hydroxylation?

The contradictory findings regarding UbiB's role in C5-hydroxylation can be addressed through several experimental approaches:

• Combined Genetic and Biochemical Analysis: Creating precise gene deletions of ubiB, ubiI, and double mutants, followed by comprehensive analysis of accumulated intermediates can help definitively assign functions. Recent research has shown that UbiI (formerly visC) functions in the C5-hydroxylation reaction, challenging earlier assumptions about UbiB's role .

• Intermediate Analysis by LC-MS/MS: Quantitative analysis of CoQ biosynthetic intermediates in various mutant strains using liquid chromatography-tandem mass spectrometry. This approach allows for precise identification of which intermediates accumulate in specific genetic backgrounds. For example, comparing the metabolite profiles of ΔubiB and ΔubiI strains would clarify their respective roles.

• In vitro Reconstitution Assays: Purification of recombinant UbiB and UbiI proteins for in vitro activity assays with potential substrates. This direct biochemical approach can determine which protein actually catalyzes the C5-hydroxylation reaction.

• Oxygen-18 Labeling Experiments: Using ¹⁸O₂ or H₂¹⁸O to track the source of oxygen atoms incorporated during hydroxylation reactions, similar to techniques used to study UbiUV-dependent hydroxylation .

• Structural Biology Approaches: Determining the crystal structures of both UbiB and UbiI would provide insights into potential catalytic mechanisms and substrate binding. The structural data for UbiI is already available and represents the first crystal structure of a monooxygenase involved in Q biosynthesis .

How does the potential protein kinase function of UbiB relate to CoQ biosynthesis?

The classification of UbiB as a member of a predicted protein kinase family suggests a regulatory role rather than direct catalytic involvement in hydroxylation reactions. Several experimental approaches can elucidate this relationship:

• Kinase Activity Assays: Testing recombinant UbiB for kinase activity using potential protein substrates involved in CoQ biosynthesis. This would determine whether UbiB phosphorylates other Ubi proteins to regulate their activity.

• Phosphoproteomic Analysis: Comparing the phosphorylation profiles of CoQ biosynthetic enzymes in wild-type and ΔubiB strains to identify differentially phosphorylated targets.

• Site-Directed Mutagenesis: Creating mutations in the kinase motifs of UbiB to determine whether they affect CoQ synthesis. If UbiB functions as a kinase, mutations in catalytic residues should disrupt CoQ production similarly to a complete gene deletion.

• Protein-Protein Interaction Studies: Using co-immunoprecipitation, bacterial two-hybrid analysis, or crosslinking approaches to identify proteins that physically interact with UbiB, providing clues about its regulatory targets.

The relationship between UbiB and the S. cerevisiae ABC1 gene provides additional context, as ABC1 is known to function as a regulatory kinase in mitochondria . Understanding the evolutionary conservation of this kinase function across different organisms would provide insights into UbiB's fundamental role in CoQ biosynthesis regulation.

What is the relationship between UbiB and oxygen-dependent versus oxygen-independent pathways of CoQ biosynthesis?

The relationship between UbiB and oxygen-dependent versus oxygen-independent pathways of CoQ biosynthesis requires careful investigation, especially in light of recent discoveries about anaerobic UQ synthesis:

• Oxygen Dependency Analysis: Research has established the existence of an anaerobic O₂-independent UQ biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes in E. coli . Determining how UbiB functions in relation to these oxygen-independent systems would clarify its role under varying oxygen conditions.

• Transcriptional Regulation Studies: The anaerobic UQ synthesis pathway genes (ubiTUV) are under the control of the O₂-sensing Fnr transcriptional regulator . Investigating whether ubiB expression is similarly regulated by oxygen levels or respiratory conditions would provide insights into its contextual function.

• Comparative Analysis Table: The following table summarizes key differences between oxygen-dependent and oxygen-independent UQ biosynthesis components:

• ¹⁸O Labeling Experiments: Using isotopic labeling to determine whether UbiB-dependent hydroxylation incorporates oxygen from O₂ (typical of monooxygenases) or from water (indicating an alternative mechanism) .

• Phenotypic Analysis Under Varying Oxygen Conditions: Comparing the growth and CoQ content of wild-type and ΔubiB strains under aerobic, microaerobic, and anaerobic conditions would reveal condition-specific dependencies on UbiB function.

Understanding the interplay between UbiB and the recently characterized UbiUVT system would provide a more comprehensive picture of how E. coli adjusts its metabolism in response to changing oxygen levels and respiratory conditions .

What are the methodological challenges in studying UbiB interactions within the larger CoQ biosynthetic complex?

Studying UbiB interactions within the larger CoQ biosynthetic complex presents several methodological challenges:

• Membrane Protein Complexes: Ubiquinone biosynthesis occurs at the membrane, and many Ubi proteins, including UbiB, are associated with or embedded in membranes. This creates challenges for:

  • Protein purification while maintaining native interactions

  • Reconstitution of multi-protein complexes in vitro

  • Crystallization for structural studies

• Transient Interactions: Many enzyme-enzyme interactions in biosynthetic pathways are transient and difficult to capture using traditional protein-protein interaction methods. Advanced approaches needed include:

  • Chemical crosslinking coupled with mass spectrometry

  • Proximity labeling techniques (BioID, APEX)

  • Single-molecule fluorescence techniques to detect short-lived interactions

• Redundancy and Compensation: The existence of parallel pathways for CoQ biosynthesis under different oxygen conditions (e.g., UbiUVT for anaerobic conditions) makes it difficult to interpret phenotypes of single gene knockouts. Researchers should consider:

  • Creating multiple gene deletions to eliminate compensatory pathways

  • Using conditional expression systems to control protein levels

  • Performing studies under carefully controlled oxygen concentrations

• Complex Operon Structure: The organization of ubiB in an operon with ubiE and yigP means that genetic manipulations might have polar effects on other genes. Methods to address this include:

  • Using CRISPR-Cas9 for precise, scarless genetic modifications

  • Complementation with individually expressed genes

  • Ribosome binding site modifications to control relative expression levels

• Post-translational Regulation: If UbiB functions as a protein kinase as suggested by sequence homology , studying its regulatory effects requires specialized techniques:

  • Phosphoproteomic analysis under various conditions

  • In vitro kinase assays with purified components

  • Generation of phosphomimetic and phospho-null mutations in target proteins

How can advanced analytical techniques be optimized for detecting UbiB-dependent intermediates in the CoQ pathway?

Advanced analytical techniques for detecting UbiB-dependent intermediates require specific optimizations:

• Sample Preparation Protocols:

  • Rapid quenching of metabolism using cold methanol or similar approaches to capture unstable intermediates

  • Extraction methods optimized for hydrophobic prenylated compounds (e.g., modified Bligh-Dyer extraction with acidification)

  • Subcellular fractionation to enrich membrane-associated intermediates

• LC-MS/MS Method Development:

  • Reverse-phase chromatography with C18 or C8 columns for separation of prenylated intermediates

  • Multiple reaction monitoring (MRM) for targeted detection of predicted intermediates

  • High-resolution accurate mass spectrometry for untargeted metabolomics to discover unexpected intermediates

• Isotopic Labeling Strategies:

  • ¹³C-labeled precursors (e.g., ¹³C-4-hydroxybenzoic acid) to trace carbon flow through the pathway

  • ¹⁸O₂ labeling to distinguish oxygen incorporation mechanisms

  • Deuterated precursors to improve detection sensitivity through isotope shifts

• Comparative Analysis Framework:

  • Parallel analysis of multiple ubi mutants (ΔubiB, ΔubiH, ΔubiF, ΔubiI, etc.)

  • Time-course experiments following induction of CoQ biosynthesis

  • Analysis under varying oxygen tensions to distinguish aerobic vs. anaerobic pathways

• Data Analysis Approaches:

  • Multivariate statistical methods (PCA, OPLS-DA) to identify pattern differences between strains

  • Pathway enrichment analysis to identify affected metabolic networks beyond CoQ

  • Integration with transcriptomic and proteomic data for systems-level understanding

These specialized approaches enable researchers to capture and characterize the specific intermediates that accumulate when UbiB function is compromised, providing mechanistic insights into its precise role in CoQ biosynthesis.