Recombinant Bartonella quintana Pantothenate synthetase (panC)

What is Bartonella quintana Pantothenate synthetase (panC) and what is its role in bacterial metabolism?

Pantothenate synthetase (PanC) catalyzes the Mg²⁺- and ATP-dependent condensation of pantoate and β-alanine to form pantothenate (vitamin B5), which is an essential precursor for coenzyme A (CoA) biosynthesis. The reaction proceeds in two sequential steps: first, activation of pantoate's carboxylic acid through adenylylation, followed by nucleophilic attack by β-alanine's amine, resulting in the release of AMP and the pantothenate product .

In Bartonella quintana, PanC is part of the essential pathway for de novo pantothenate biosynthesis. B. quintana has a compact genome of approximately 1,581,384 bp , and like other pathogenic bacteria, it relies on its own biosynthesis of pantothenate for optimal fitness and virulence, particularly within host environments where pantothenate acquisition may be limited.

How does the genomic context of panC in Bartonella quintana compare to other bacterial species?

The panC gene is one of approximately 1,143 protein-coding genes identified in the B. quintana genome . Compared to the closely related species B. henselae (which has 1,491 genes), B. quintana exhibits a more streamlined genome that has undergone reductive evolution. This genomic reduction is characteristic of host-adapted pathogens and reflects the specialized lifestyle of B. quintana as a human-specific pathogen.

The B. quintana genome shows strong strand-specific mutation biases, with large excesses of G and T on the leading strands, a feature that influences the codon usage and potentially the expression levels of genes including panC . Understanding this genomic context is important when designing expression constructs for recombinant protein production.

What are the structural characteristics of Pantothenate synthetase that influence its function?

While B. quintana PanC has not been specifically characterized structurally in the provided literature, inferences can be made from well-studied homologs. PanC enzymes typically function as dimers and contain:

• A nucleotide-binding domain for ATP

• A substrate-binding pocket specific for pantoate

• A binding site for β-alanine that becomes accessible after adenylylation of pantoate

• Mg²⁺ binding sites that coordinate ATP positioning

These structural features facilitate the two-step reaction mechanism. Based on studies with Mycobacterium tuberculosis PanC (MtPanC), the enzyme has been crystallized in various forms including apo, as well as in complex with substrates, inhibitors, and reaction intermediates, revealing important mechanistic details about the catalytic process .

What is the optimal protocol for cloning, expressing, and purifying recombinant B. quintana PanC?

Based on successful approaches with other recombinant Bartonella proteins, the following methodology is recommended:

• Gene amplification and cloning:

  • PCR-amplify the panC gene from B. quintana genomic DNA

  • Clone into pET200D/TOPO or similar expression vector

  • Confirm insertion by Sanger sequencing

• Expression:

  • Transform E. coli BL21(DE3) with the recombinant plasmid

  • Grow cultures at 37°C to mid-log phase (OD₆₀₀ = 0.6-0.8)

  • Induce with IPTG (0.5-1.0 mM) and continue expression at lower temperatures (16-25°C) to enhance solubility

  • Harvest cells after 4-16 hours by centrifugation

• Purification:

  • Resuspend cell pellet in buffer containing protease inhibitors

  • Lyse cells using sonication or mechanical disruption

  • Clarify lysate by centrifugation

  • Purify using Ni-NTA affinity chromatography (for His-tagged protein)

  • Further purify by ion exchange and/or size exclusion chromatography

  • Confirm purity by SDS-PAGE and Western blot analysis

How can the enzymatic activity of recombinant B. quintana PanC be measured?

The standard assay for PanC activity couples the production of AMP to the oxidation of NADH:

• Coupled enzymatic assay:

  • Reaction mixture contains purified PanC, pantoate, β-alanine, ATP, Mg²⁺

  • AMP produced is coupled to NADH oxidation via auxiliary enzymes (myokinase, pyruvate kinase, lactate dehydrogenase)

  • Monitor decrease in NADH absorbance at 340 nm

  • Calculate activity using extinction coefficient of NADH (6220 M⁻¹cm⁻¹)

• Direct monitoring of product formation:

  • HPLC or LC-MS methods to detect pantothenate formation

  • Radioactive assays using ¹⁴C-labeled substrates

For kinetic characterization, varying substrate concentrations should be used to determine parameters such as Kₘ and kcat. Based on MtPanC studies, expected Kₘ values might be in the ranges: Kₘ(pantoate) ~130 μM, Kₘ(β-Ala) ~800 μM, Kₘ(ATP) ~2.6 mM .

What methods can be used to characterize the structure of recombinant B. quintana PanC?

Structural characterization typically employs multiple complementary techniques:

How can structure-based approaches be used to design inhibitors of B. quintana PanC?

Structure-based inhibitor design for B. quintana PanC would involve:

• Virtual screening:

  • Generate homology model based on crystal structures of homologous PanC enzymes

  • Identify binding pockets and key residues involved in catalysis

  • Screen virtual libraries against these sites using docking algorithms

• Fragment-based approaches:

  • Screen fragment libraries for weak binders

  • Elaborate fragments to improve potency and selectivity

  • Link fragments that bind to adjacent pockets

• Mechanism-based inhibitors:

  • Design transition-state analogs that mimic the pantoyl adenylate intermediate

  • Develop covalent inhibitors that target catalytic residues

• Rational optimization:

  • Use structure-activity relationship (SAR) studies to optimize hits

  • Improve pharmacokinetic properties while maintaining target engagement

Since PanC catalyzes a two-step reaction, inhibitors can be designed to interfere with either the adenylylation step or the subsequent nucleophilic attack .

What are the challenges in developing selective inhibitors of B. quintana PanC?

Several challenges must be addressed when developing selective inhibitors:

• Selectivity over human enzymes:

  • Pantothenate is an essential vitamin for humans, but humans lack the biosynthetic pathway

  • Focus on structural features unique to bacterial PanC

• Specificity among bacterial species:

  • Consider conservation of catalytic residues across bacterial PanC enzymes

  • Target regions with sequence/structural differences unique to B. quintana

• Physicochemical properties:

  • Design compounds that can penetrate bacterial cell membranes

  • Consider efflux mechanisms that might limit intracellular concentration

• Resistance development:

  • Map potential resistance mutations

  • Design inhibitors with high barriers to resistance

How can site-directed mutagenesis be used to investigate the catalytic mechanism of B. quintana PanC?

Site-directed mutagenesis studies would typically follow this approach:

• Target residue identification:

  • Perform sequence alignments with well-characterized PanC enzymes

  • Identify conserved residues likely involved in catalysis

  • Focus on residues in the active site, substrate binding regions, and dimer interface

• Mutagenesis strategy:

  • Introduce conservative mutations (e.g., Asp→Glu) to probe function

  • Create alanine substitutions to eliminate side chain contributions

  • Design mutations that alter substrate specificity

• Functional characterization:

  • Compare kinetic parameters (Kₘ, kcat) of mutants with wild-type enzyme

  • Analyze effects on substrate binding using isothermal titration calorimetry (ITC)

  • Determine structural changes using X-ray crystallography

• Mechanistic insights:

  • Correlate kinetic changes with structural alterations

  • Develop refined models of the catalytic mechanism

How do the kinetic parameters of B. quintana PanC compare with PanC from other bacterial species?

While specific kinetic parameters for B. quintana PanC are not provided in the search results, a comparative analysis with well-characterized PanC enzymes helps establish expectations:

Table 1: Comparative Kinetic Parameters of Pantothenate Synthetase from Various Bacterial Species

*Values for E. coli are presented as ranges from literature reports not included in the search results.

When investigating B. quintana PanC, researchers should anticipate kinetic parameters potentially in these ranges, though evolutionary adaptations specific to B. quintana's lifestyle might result in distinct kinetic properties that reflect its host-adapted status and genome reduction .

What functional genomics approaches can be used to validate the essentiality of panC in B. quintana?

Several complementary approaches can assess the essentiality of panC:

• Conditional knockout strategies:

  • Generate inducible expression systems where panC expression can be controlled

  • Monitor growth and viability under varying expression levels

• Genetic complementation:

  • Create pantothenate auxotrophic strains with disrupted panC

  • Test rescue with exogenous pantothenate or genetic complementation

• Transposon mutagenesis:

  • Perform saturating transposon mutagenesis

  • Identify regions where transposon insertions are not recovered (essential genes)

• CRISPRi approaches:

  • Use dCas9-based interference to repress panC expression

  • Quantify growth effects under varying repression conditions

The critical role of pantothenate in CoA biosynthesis, which is essential for numerous cellular processes, suggests that panC would likely be essential under conditions where exogenous pantothenate is limited, similar to findings in other bacterial pathogens .

How does recombinant B. quintana PanC activity respond to various environmental conditions relevant to host infection?

Understanding environmental regulation helps illuminate the enzyme's role during infection:

Table 2: Environmental Factors Affecting Pantothenate Synthetase Activity

These investigations would help characterize the adaptations of B. quintana PanC to its human host environment and inform strategies for therapeutic targeting.

What are promising approaches for developing PanC-targeted therapeutics against Bartonella infections?

Several strategies show promise for targeting PanC in future therapeutic development:

• Structure-based drug design:

  • Leverage structural information to design competitive inhibitors

  • Develop transition-state analogs specific to the adenylylation mechanism

• Antimetabolite approaches:

  • Create pantothenate analogs that can be processed by bacterial PanK but block downstream metabolism

  • Design pro-drugs that are activated by bacterial systems

• Combination strategies:

  • Pair PanC inhibitors with compounds targeting other steps in the CoA pathway

  • Combine with conventional antibiotics to enhance efficacy

• Alternative delivery systems:

  • Develop nanoparticle formulations for improved delivery to infection sites

  • Create prodrug approaches that target bacteria specifically

• Repurposing existing compounds:

  • Screen FDA-approved drug libraries for PanC inhibitory activity

  • Modify existing scaffolds known to target related ATP-utilizing enzymes

The essential nature of pantothenate biosynthesis for B. quintana virulence makes PanC an attractive target, particularly given the absence of this pathway in humans .

How might systems biology approaches enhance our understanding of pantothenate metabolism in B. quintana?

Integrative approaches can provide deeper insights into pantothenate metabolism: