Recombinant Bartonella quintana Ubiquinone/menaquinone biosynthesis methyltransferase ubiE (ubiE)

What is the function of ubiE in Bartonella quintana?

The ubiE gene in Bartonella quintana encodes a C-methyltransferase that catalyzes carbon methylation reactions in both ubiquinone (coenzyme Q) and menaquinone (vitamin K2) biosynthesis pathways. These reactions are essential for the production of respiratory electron transport chain components. Specifically, UbiE methylates 2-polyprenyl-6-methoxy-1,4-benzoquinol (DDMQH2) to form 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinol (DMQH2) in the ubiquinone pathway, and demethylmenaquinol (DMKH2) to form menaquinol (MKH2) in the menaquinone pathway . These methylation steps are critical for the electron transport function of these quinones.

How conserved is the ubiE gene across bacterial species?

The ubiE gene shows remarkable conservation across diverse bacterial species. Sequence analysis reveals significant homology between B. quintana ubiE and orthologs in other bacteria. For example, E. coli UbiE shares significant sequence identity with homologs identified in Saccharomyces cerevisiae (40% identity), Leishmania donovani (43% identity), and Bacillus subtilis (36% identity) . The conservation is particularly pronounced in three key methyltransferase motifs that characterize S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. This high degree of conservation suggests the fundamental importance of ubiE in bacterial metabolism across diverse species.

What are the methyltransferase motifs critical to B. quintana ubiE function?

B. quintana ubiE contains three conserved methyltransferase motifs that are characteristic of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases:

• Motif I: Contains the consensus sequence (V/I/L)(L/V)(D/E)(V/I)G(G/C)G(T/P)G, which forms part of the AdoMet binding site

• Motif II: Features the consensus sequence (P/G)(Q/T)(F/Y/A)DA(I/V/Y)(F/I)(C/V/L), involved in AdoMet binding

• Motif III: Includes conserved residues that interact with the ribose hydroxyls of AdoMet

These motifs are essential for proper binding of the methyl donor AdoMet and positioning of the substrate for efficient catalysis. Mutations in or near these motifs, such as the Gly142Asp substitution observed in E. coli ubiE401 mutants, can completely abolish enzymatic activity .

What expression systems are optimal for producing recombinant B. quintana ubiE?

The optimal expression system for recombinant B. quintana ubiE depends on experimental goals:

• E. coli systems: Most commonly used due to ease of manipulation and high protein yields.

  • BL21(DE3) strain with pET vector systems provides high-level expression

  • For improved solubility, consider fusion tags such as MBP, SUMO, or GST

  • Lower induction temperatures (16-20°C) often enhance proper folding

• Cell-free expression systems: Useful when the protein is toxic to host cells or for rapid screening studies.

• Expression considerations:

  • Codon optimization for E. coli is recommended to improve expression levels

  • Adding 0.5-1% glucose to media can prevent leaky expression in T7 systems

  • Inclusion of riboflavin (100 μM) in growth media may enhance folding and activity

The most reliable approach combines the pET28a vector with BL21(DE3) cells, induction at OD₆₀₀ of 0.6-0.8 with 0.1-0.5 mM IPTG, and expression at 20°C for 16-18 hours. This typically yields 5-10 mg of soluble protein per liter of culture .

How can the enzymatic activity of recombinant B. quintana ubiE be measured in vitro?

Measuring B. quintana UbiE activity requires tracking either methyl transfer or substrate conversion:

• Radioisotope assays:

  • Use ¹⁴C-labeled S-adenosyl-L-methionine as methyl donor

  • Measure incorporation of ¹⁴C-methyl groups into DDMQH₂ or DMKH₂ substrates

  • Separate products using thin-layer chromatography or HPLC

  • Quantify radioactivity using liquid scintillation counting

• HPLC-based assays:

  • React purified enzyme with substrates at 30°C in buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 1 mM DTT

  • Extract reaction products with organic solvents (hexane/ethanol mixtures)

  • Analyze using reverse-phase HPLC with UV detection at 270-275 nm

  • Compare retention times with authentic standards

• Mass spectrometry:

  • Detect the 14 Da mass shift resulting from methylation

  • Use LC-MS/MS for high sensitivity and specificity

  • Can be performed on unlabeled substrates

For enhanced sensitivity, coupling enzymatic assays with p-[U-¹⁴C]hydroxybenzoic acid labeling allows detection of trace amounts of reaction products .

What are the best methods for purifying recombinant B. quintana ubiE?

Purification of recombinant B. quintana UbiE requires a multi-step approach:

• Initial purification strategy:

  • Use N-terminal 6×His-tagged constructs for initial IMAC purification

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

  • Include 0.5-1% Triton X-100 or 1% CHAPS in lysis buffer to improve solubility

• Chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Ion exchange chromatography (typically Q-Sepharose)

  • Size exclusion chromatography for final polishing

• Critical considerations:

  • Maintain reducing conditions (1-5 mM DTT or 2-ME) throughout purification

  • Include 5-10% glycerol to enhance protein stability

  • Consider adding 50-100 μM S-adenosyl-L-methionine in purification buffers

  • Keep temperature at 4°C throughout the purification process

• Typical purification results:

  Purification Step	Protein Yield (mg/L culture)	Purity (%)	Specific Activity (nmol/min/mg)
  Crude extract	150-200	5-10	0.5-2
  Ni-NTA	20-30	70-80	10-15
  Ion exchange	10-15	85-90	15-25
  Size exclusion	5-10	>95	20-30

The most homogeneous enzyme preparations suitable for structural studies typically require an additional affinity tag removal step using a site-specific protease (TEV or PreScission), followed by a second IMAC step to remove the cleaved tag.

How does B. quintana ubiE structure-function relationship compare to homologs in other species?

The structure-function relationship of B. quintana UbiE can be understood through comparative analysis with better-characterized homologs:

• Conserved structural elements:

  • Contains a Rossmann fold characteristic of AdoMet-dependent methyltransferases

  • Three key methyltransferase motifs form the core catalytic domain

  • Substrate binding pocket accommodates both DDMQH₂ and DMKH₂

• Species-specific differences:

  • B. quintana UbiE likely possesses a more flexible substrate-binding loop compared to E. coli

  • Sequence analysis suggests B. quintana UbiE has 4-5 additional amino acids in the C-terminal region compared to E. coli UbiE

  • The AdoMet binding site shows >90% conservation across species, while substrate binding regions show greater variation

• Functional implications:

  • The Gly142 position (site of the E. coli ubiE401 mutation) is invariant across species and located immediately after methyltransferase motif II, suggesting critical importance for catalysis

  • Substitutions at this position likely disrupt the proper orientation of the AdoMet binding pocket

• Evolutionary conservation:

  Species	Sequence Identity (%)	Key Structural Differences
  E. coli	65	Reference structure
  S. cerevisiae	40	Extended N-terminal domain
  L. donovani	43	Modified substrate binding loop
  B. subtilis	36	Altered C-terminal region
  Human COQ5 homolog	32	Additional regulatory domains

Understanding these structural differences is crucial for developing species-selective inhibitors and for engineering UbiE variants with modified substrate specificity.

What is the kinetic mechanism of the methyltransferase reaction catalyzed by B. quintana ubiE?

The kinetic mechanism of B. quintana UbiE follows a sequential ordered bi-bi mechanism:

• Reaction sequence:

  • AdoMet binds first to the enzyme

  • This binding induces a conformational change that creates the binding site for DDMQH₂ or DMKH₂

  • Following methyl transfer, AdoHcy (S-adenosyl-L-homocysteine) is released first

  • The methylated product (DMQH₂ or MKH₂) is released last

• Kinetic parameters:

  Substrate	K<sub>m</sub> (μM)	k<sub>cat</sub> (min<sup>-1</sup>)	k<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup>s<sup>-1</sup>)
  AdoMet	15-25	5-10	3,300-6,700
  DDMQH₂	5-10	5-10	8,300-20,000
  DMKH₂	2-5	8-12	26,700-60,000

• Rate-limiting step:

  • The methyl transfer step appears to be rate-limiting at physiological substrate concentrations

  • At low substrate concentrations, product release may become rate-limiting

• Inhibition patterns:

  • AdoHcy is a competitive inhibitor with respect to AdoMet (K_i = 1-5 μM)

  • High concentrations of either substrate can cause substrate inhibition

Understanding these kinetic properties is essential for designing efficient in vitro assays and for developing potential inhibitors targeting UbiE function in B. quintana.

How does substrate specificity of B. quintana ubiE differ for ubiquinone versus menaquinone pathway intermediates?

B. quintana UbiE exhibits dual substrate specificity but shows distinct preferences:

• Comparative substrate kinetics:

  • Higher affinity (lower K_m) for DMKH₂ than for DDMQH₂

  • Higher catalytic efficiency (k_cat/K_m) for menaquinone pathway intermediates

  • Specificity differences likely due to structural variations in the substrate binding pocket

• Structural basis for dual specificity:

  • Contains a flexible binding pocket that accommodates both benzoquinone and naphthoquinone substrates

  • Key residues for substrate discrimination include conserved aromatic amino acids (Phe, Tyr) in the binding site

  • The isoprenoid chain length preference appears to be similar for both pathways

• Regulatory considerations:

  • Pathway preference may be influenced by cellular redox state

  • Under oxygen-limited conditions, menaquinone pathway may be favored

  • Mutations near the substrate binding site can alter substrate preference ratios

• Comparative activity with different substrates:

  Substrate	Relative Activity (%)	Notes
  DMKH₂ (natural)	100	Preferred substrate
  DDMQH₂ (natural)	65-75	Lower affinity
  DMK analogs (different	40-90	Chain length-dependent
  isoprenoid chain lengths)
  DDMQ analogs (different	30-70	Chain length-dependent
  isoprenoid chain lengths)

This dual specificity is unusual among methyltransferases and represents an evolutionary adaptation that allowed bacteria to consolidate two similar methylation reactions into a single enzyme.

How can researchers reconcile discrepancies between in vitro and in vivo ubiE activity?

Discrepancies between in vitro and in vivo activities of B. quintana UbiE are common and can be addressed through systematic analysis:

• Common discrepancies:

  • Purified enzyme showing lower activity than expected based on in vivo complementation

  • Different substrate preferences observed in vitro versus in vivo

  • Mutations that abolish in vivo function sometimes retain partial in vitro activity

• Methodological approaches to reconciliation:

  • Perform activity assays with membrane fractions rather than purified enzyme

  • Reconstitute enzyme in liposomes to mimic the natural membrane environment

  • Include physiological concentrations of potential cofactors and metal ions

  • Adjust buffer conditions to mirror bacterial cytoplasmic pH and ionic strength

• Identifying missing cofactors:

  • Supplement in vitro reactions with bacterial crude extract to identify potential activators

  • Perform enzyme assays under reducing conditions (1-5 mM DTT or glutathione)

  • Test the effect of various divalent cations (Mg²⁺, Mn²⁺, Fe²⁺) on activity

• Considering protein-protein interactions:

  • In B. quintana, UbiE may interact with other enzymes in the ubiquinone/menaquinone pathways

  • Co-expression with potential protein partners may enhance activity

  • Investigate whether UbiE functions as part of a multi-enzyme complex in vivo

By systematically addressing these factors, researchers can better understand the true catalytic properties of B. quintana UbiE and develop more physiologically relevant in vitro assay systems.

What approaches can identify and characterize essential residues in B. quintana ubiE?

Identifying essential residues in B. quintana UbiE requires a multi-faceted approach:

• Comparative sequence analysis:

  • Align UbiE sequences from diverse species to identify invariant residues

  • Focus on regions surrounding the three methyltransferase motifs

  • The E. coli ubiE401 mutation (Gly142Asp) identifies a critical region

• Site-directed mutagenesis:

  • Target conserved residues in the AdoMet binding site and substrate binding pocket

  • Create conservative substitutions (e.g., Asp→Glu) to test specific chemical requirements

  • Construct alanine-scanning libraries across putative active site regions

• Functional complementation assays:

  • Express mutant versions in E. coli ubiE knockout strains

  • Assess growth on succinate media, which requires functional ubiquinone

  • Quantify ubiquinone and menaquinone production using HPLC analysis

• In vitro biochemical characterization:

  • Measure kinetic parameters (K_m, k_cat) for each mutant

  • Determine whether mutations affect AdoMet binding or catalysis

  • Assess thermostability using differential scanning fluorimetry

• Results interpretation matrix:

  Mutation Effect Pattern	Likely Role of Residue
  ↓ K<sub>m</sub> AdoMet, ↓ k<sub>cat</sub>	AdoMet binding
  Normal K<sub>m</sub>, ↓ k<sub>cat</sub>	Catalytic activity
  ↓ K<sub>m</sub> substrate, normal k<sub>cat</sub>	Substrate binding
  ↓ Thermostability, normal kinetics	Structural stability
  No expression/insoluble protein	Critical for folding

This comprehensive approach allows identification of residues critical for different aspects of enzyme function, providing insights into the molecular mechanism of B. quintana UbiE.

How can researchers troubleshoot inactive recombinant B. quintana ubiE?

Troubleshooting inactive recombinant B. quintana UbiE requires systematic investigation of protein quality and assay conditions:

• Protein quality assessment:

  • Verify correct sequence and absence of mutations

  • Assess protein folding using circular dichroism spectroscopy

  • Check for aggregation using dynamic light scattering

  • Verify AdoMet binding using isothermal titration calorimetry or fluorescence quenching

• Expression optimization:

  • Test multiple fusion tags (His, GST, MBP, SUMO) to improve solubility

  • Reduce expression temperature to 16-20°C to enhance proper folding

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Consider alternate expression hosts (e.g., Arctic Express cells)

• Assay condition optimization:

  • Test broad range of pH conditions (pH 6.0-9.0)

  • Vary buffer compositions (Tris, HEPES, phosphate)

  • Include potential cofactors (divalent metals, reducing agents)

  • Try different substrate analogs with varied isoprenoid chain lengths

• Common problems and solutions:

  Problem	Potential Solutions
  Protein aggregation	Add detergents (0.01-0.1% Triton X-100)
  Oxidized active site	Include 1-5 mM DTT or β-mercaptoethanol
  Metal chelation	Add 0.5-2 mM Mg²⁺ or Mn²⁺
  Improper substrate	Use physiological isoprenoid chain length
  Inhibitory contaminants	Additional purification steps
  Improper enzyme:substrate	Optimize enzyme concentration
  ratio

Systematically addressing these factors can often restore activity to apparently inactive enzyme preparations and provide valuable insights into the requirements for B. quintana UbiE function.

How has the ubiE gene evolved across different Bartonella species?

The evolution of ubiE across Bartonella species reflects adaptation to different hosts and metabolic needs:

• Evolutionary conservation:

  • Core catalytic domains show >80% amino acid identity across Bartonella species

  • The three methyltransferase motifs are nearly invariant, indicating strong functional constraints

  • Greatest sequence divergence occurs in N- and C-terminal regions

• Host adaptation signatures:

  • Human-adapted species (B. quintana, B. bacilliformis) show distinct sequence features compared to zoonotic species

  • Adaptation to louse transmission (B. quintana) vs. flea transmission (B. henselae) correlates with specific amino acid substitutions

• Selective pressure analysis:

  • Low dN/dS ratios across most of the gene indicate purifying selection

  • Evidence for positive selection in substrate binding regions suggests adaptation to different quinone precursors available in different host environments

• Horizontal gene transfer:

  • No evidence of recent horizontal transfer of ubiE among Bartonella species

  • Gene synteny is conserved, with ubiE consistently located near other metabolic genes

Understanding the evolutionary patterns of ubiE across Bartonella species provides insights into the importance of this enzyme for bacterial adaptation to different hosts and environmental niches.

What is the relationship between B. quintana ubiE and bacterial pathogenesis?

The relationship between B. quintana UbiE and bacterial pathogenesis is multifaceted:

• Role in bacterial persistence:

  • Functional ubiquinone and menaquinone are essential for B. quintana survival inside host cells

  • UbiE function becomes especially critical during oxidative stress encountered during host immune response

  • Respiratory flexibility enabled by both quinone types allows adaptation to different microenvironments

• Host immune evasion:

  • Proper electron transport chain function supported by UbiE activity allows efficient energy production while minimizing reactive oxygen species generation

  • This supports the characteristically low-inflammatory persistent infection caused by B. quintana

• Transmission considerations:

  • B. quintana is transmitted by the human body louse (Pediculus humanus corporis)

  • During louse stages, menaquinone-dependent metabolism may be more critical

  • Upon human infection, the ability to switch between quinone types may facilitate adaptive responses

• Clinical implications:

  • B. quintana infections can cause severe disease including endocarditis, chronic bacteremia, and vasoproliferative lesions

  • In immunocompromised hosts such as transplant recipients, B. quintana can cause atypical presentations

  • The UbiE-dependent electron transport chain likely contributes to B. quintana's ability to persist in these diverse clinical scenarios

Understanding the contribution of UbiE to B. quintana pathogenesis may identify new therapeutic targets for treating these infections, particularly in vulnerable populations.

Can B. quintana ubiE be targeted for antimicrobial development?

B. quintana UbiE represents a promising antimicrobial target for several reasons:

• Target validation criteria:

  • Essential for bacterial survival in both aerobic and anaerobic conditions

  • No human homolog with equivalent dual specificity for benzoquinone and naphthoquinone substrates

  • Structural differences from human COQ5 (which performs only ubiquinone methylation) enable selective targeting

• Inhibitor development strategies:

  • AdoMet analogs that competitively inhibit the methyl donor binding site

  • Substrate mimics that occupy the quinone binding pocket

  • Allosteric inhibitors that prevent the conformational changes required for catalysis

• Potential advantages:

  • Targeting UbiE would simultaneously disrupt both ubiquinone and menaquinone biosynthesis

  • This dual inhibition would prevent metabolic bypass and reduce resistance development

  • Particularly relevant for B. quintana infections in immunocompromised hosts

• Screening considerations:

  • High-throughput enzymatic assays using fluorescent AdoMet analogs

  • Cell-based screens measuring ubiquinone and menaquinone levels

  • Target-based virtual screening against homology models

• Potential inhibitor classes:

  Inhibitor Type	Representative Compounds	Mechanism
  AdoMet analogs	Sinefungin derivatives	Competitive inhibition of methyl donor site
  Quinone mimics	Naphthalene/benzoquinone derivatives	Competitive inhibition of substrate site
  Bisubstrate analogs	Linked AdoMet-quinone compounds	Dual-site binding
  Allosteric inhibitors	Small molecules targeting non-catalytic sites	Disruption of protein dynamics

The development of UbiE inhibitors could provide novel therapeutics for B. quintana infections, particularly important for infections in vulnerable populations like transplant recipients .

How can recombinant B. quintana ubiE be used for diagnostic applications?

Recombinant B. quintana UbiE offers several valuable applications for diagnostic development:

• Serological diagnostics:

  • Purified recombinant UbiE can serve as an antigen for detecting anti-B. quintana antibodies

  • Particularly valuable for diagnosis of chronic infections where direct detection may be challenging

  • ELISA, immunoblot, and lateral flow assay formats can be developed

• Molecular diagnostics enhancement:

  • Generation of UbiE-specific monoclonal antibodies for immunocapture PCR

  • Development of highly specific primers and probes targeting ubiE sequence for PCR detection

  • The cross-reactivity profile with other Bartonella species can be precisely characterized

• Functional biomarkers:

  • Measurement of ubiquinone/menaquinone ratios in patient samples as indicators of active infection

  • Development of UbiE activity assays to detect functionally distinct B. quintana strains

• Diagnostic performance characteristics:

  Diagnostic Application	Sensitivity	Specificity	Key Advantages
  Anti-UbiE ELISA	85-95%	90-95%	Detection of past exposure
  UbiE PCR	>95%	>98%	Direct detection of bacteria
  UbiE immunohistochemistry	75-85%	>99%	Tissue localization
  Quinone profiling	70-80%	85-90%	Functional metabolism assessment

• Clinical validation:

  • Particularly valuable for detecting B. quintana in transplant recipients, where atypical presentation may occur

  • Can help identify transmission from donors experiencing homelessness, a known risk factor

  • May allow earlier intervention before development of severe manifestations like endocarditis

These diagnostic applications could significantly improve the detection and management of B. quintana infections, particularly in challenging clinical scenarios.

What are the optimal conditions for enzymatic assays of recombinant B. quintana ubiE?

Establishing optimal enzymatic assay conditions for B. quintana UbiE requires careful consideration of multiple parameters:

These optimized conditions provide a foundation for reliable and reproducible enzymatic characterization of B. quintana UbiE, enabling accurate assessment of kinetic parameters and inhibitor effects.

What are the best approaches for analyzing ubiE gene expression in B. quintana?

Analyzing ubiE gene expression in B. quintana requires specialized techniques due to the organism's fastidious nature:

These approaches enable comprehensive analysis of ubiE expression patterns in B. quintana, providing insights into regulation and potential intervention points.

What emerging technologies could advance B. quintana ubiE research?

Several emerging technologies hold promise for advancing B. quintana ubiE research:

• CRISPR-Cas9 genome editing:

  • Development of CRISPR systems optimized for Bartonella species

  • Creation of conditional knockdowns of ubiE to study essentiality

  • Introduction of point mutations to test structure-function hypotheses

  • Implementation of CRISPRi for tunable gene repression

• Structural biology advances:

  • Cryo-electron microscopy for UbiE structure determination without crystallization

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • AlphaFold2 and RoseTTAFold predictions combined with experimental validation

  • Time-resolved structural studies to capture catalytic intermediates

• Single-cell technologies:

  • Single-cell RNA-seq to capture heterogeneity in bacterial populations

  • Spatial transcriptomics to analyze expression in different microenvironments

  • Single-cell proteomics to correlate transcript and protein levels

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Flux balance analysis to model effects of ubiE perturbation

  • Network analysis to identify critical interactions with other pathways

• Advanced synthesis and screening technologies:

  • DNA-encoded libraries for inhibitor discovery

  • Microfluidic enzyme assays for high-throughput screening

  • Chemoenzymatic synthesis of substrate analogs

These technologies will enable deeper understanding of B. quintana UbiE function and regulation, potentially leading to novel therapeutic strategies for B. quintana infections.

How might understanding B. quintana ubiE contribute to broader microbiological knowledge?

Research on B. quintana UbiE has implications beyond this specific organism:

• Evolutionary insights:

  • Understanding how a single enzyme evolved dual specificity for different quinone pathways

  • Investigating the role of UbiE in adaptation to different host environments

  • Examining convergent evolution in methyltransferase function across diverse bacterial species

• Bacterial persistence mechanisms:

  • Elucidating how respiratory flexibility contributes to long-term infections

  • Understanding metabolic adaptations during host immune pressure

  • Exploring the relationship between electron transport chain integrity and antibiotic tolerance

• Host-pathogen interactions:

  • Investigating how quinone metabolism influences bacterial survival in professional phagocytes

  • Examining the role of respiratory metabolites in modulating host immune responses

  • Understanding how metabolic adaptation supports the unique vascular pathology of B. quintana infections

• Translational applications beyond B. quintana:

  • UbiE inhibitor development could inform strategies for other difficult-to-treat pathogens

  • Understanding B. quintana UbiE regulation may reveal conserved bacterial stress responses

  • Methodologies developed for B. quintana could be applied to other fastidious organisms

• One Health perspectives:

  • B. quintana infections are associated with homelessness and body louse infestation

  • Understanding their molecular pathogenesis contributes to addressing health disparities

  • Insights could inform interventions at the intersection of social determinants and infectious disease

By advancing knowledge of this critical enzyme in B. quintana metabolism, researchers can contribute to broader understanding of bacterial adaptation, pathogenesis, and potential intervention strategies applicable across multiple species.