Recombinant Acinetobacter sp. Coenzyme PQQ synthesis protein D (pqqD)

What is PqqD and how does it function in the PQQ biosynthetic pathway?

PqqD is a 10-kDa protein that functions as a peptide chaperone in the pyrroloquinoline quinone (PQQ) biosynthetic pathway. PQQ biosynthesis involves five conserved genes, pqqA-E, with PqqD being essential for PQQ production. Research has demonstrated that PqqD binds to the peptide substrate PqqA with high affinity (Kᴅ ~200 nM) and forms a crucial ternary complex with the radical S-adenosylmethionine (RS) protein PqqE . This interaction is believed to facilitate the first step in PQQ biosynthesis, which involves the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the PqqA peptide .

The functional significance of PqqD has been established through mutational studies in Acinetobacter calcoaceticus, where PQQ-deficient mutants showed inability to grow on substrates like L-arabinose, which requires the PQQ-containing glucose dehydrogenase for metabolism . The chaperone function of PqqD appears to be conserved across different bacterial species, including Methylobacterium extorquens AM1 and Klebsiella pneumoniae, suggesting its fundamental role in PQQ biosynthesis .

What experimental evidence supports PqqD's role as a peptide chaperone?

Multiple experimental approaches have confirmed PqqD's chaperone function. Surface plasmon resonance (SPR) experiments demonstrate that Methylobacterium extorquens PqqD (MePqqD) binds MePqqA with a Kᴅ of 390 ± 80 nM, while isothermal calorimetry (ITC) measurements yielded similar values (Kᴅ = 130 ± 30 nM) . Neither PqqB nor PqqE alone showed binding to PqqA in their as-isolated forms, highlighting PqqD's unique role in substrate recognition .

Native mass spectrometry experiments have captured the formation of a 1:1:1 ternary MePqqA-D-E complex, with SPR experiments determining the Kᴅ of the MePqqAD complex binding to MePqqE to be 4.5 ± 1.5 μM . This ternary complex formation suggests that PqqD functions as a molecular adapter, bringing together the peptide substrate and the RS enzyme that catalyzes the initial modification .

Cross-feeding experiments with PQQ-deficient mutants in Acinetobacter calcoaceticus failed to reconstitute PQQ synthesis, indicating that the biosynthetic intermediates remain enzyme-bound throughout the pathway, consistent with PqqD's proposed chaperone function .

How do the structural properties of PqqD relate to its function?

The structural properties of PqqD have been investigated using a combination of crystallography and solution-based techniques, revealing interesting differences between species. Small-angle X-ray scattering (SAXS) analysis of Klebsiella pneumoniae PqqD (KpPqqD) indicates that it exists as a monomer in solution with a globular structure . This contrasts with the crystal structure of Xanthomonas campestris PqqD (XcPqqD), which shows a domain-swapped dimer with each monomer containing an α-helix bundle and an extended β1-β2 hairpin feature .

This structural discrepancy suggests that PqqD may undergo conformational changes related to its function. When a model of a compact XcPqqD monomer was generated using the β1 and β2 strands from one polypeptide chain and the β3, α1, α2, and α3 motifs from a second chain, its theoretical SAXS profile matched the experimental profile for KpPqqD . This supports the hypothesis that PqqD's structure is dynamic and may adopt different conformations during its chaperone function, potentially facilitating tight complex formation with PqqA .

What methodologies are most effective for characterizing PqqD-peptide interactions?

Characterizing PqqD-peptide interactions requires a multi-technique approach to obtain comprehensive binding data. The most effective methodologies include:

Surface Plasmon Resonance (SPR): This technique allows real-time monitoring of binding events and determination of kinetic parameters. For MePqqD-MePqqA interactions, SPR experiments revealed a Kᴅ of 390 ± 80 nM . Dual injection SPR techniques can also assess ternary complex formation, as demonstrated for the MePqqAD-MePqqE interaction (Kᴅ = 4.5 ± 1.5 μM) .

Isothermal Titration Calorimetry (ITC): This technique provides direct measurement of binding thermodynamics. ITC experiments confirmed SPR results for MePqqD-MePqqA binding (Kᴅ = 130 ± 30 nM) and established a 1:1 molar ratio . The following table summarizes binding affinities determined by different methods:

Native Mass Spectrometry: This technique confirms complex formation and stoichiometry under near-native conditions. It successfully detected the 1:1:1 ternary MePqqA-D-E complex, providing direct evidence for the proposed chaperone mechanism .

For optimal results, researchers should employ at least two complementary techniques when characterizing PqqD interactions, as each provides unique insights into binding parameters and complex formation.

How does the oligomerization state of PqqD impact its function in different bacterial species?

The oligomerization state of PqqD varies between bacterial species and appears to impact its functional properties. Analytical size exclusion chromatography (SEC) and SAXS analyses indicate that KpPqqD exists primarily as a monomer in solution, contrasting with the domain-swapped dimer observed in the XcPqqD crystal structure . This discrepancy raises important questions about PqqD's native state and functional mechanism.

SAXS data for KpPqqD yielded an experimental radius of gyration (Rg) of 15.5 Å and a maximum particle dimension (Dmax) of 50 Å, consistent with a monomeric, globular structure . When compared to the theoretical parameters calculated from the XcPqqD crystal structure, significant differences were observed, suggesting that the crystallization conditions may have induced an artificial oligomerization state .

Further research using techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) or crosslinking studies would help elucidate whether PqqD undergoes dynamic oligomeric transitions during its chaperone function and how these might differ across bacterial species.

What is the significance of the 1:1:1 ternary complex between PqqA, PqqD, and PqqE in the context of radical SAM chemistry?

The formation of a 1:1:1 ternary complex between PqqA, PqqD, and PqqE represents a crucial mechanistic insight into radical SAM enzyme-mediated peptide modification. PqqE belongs to the radical S-adenosylmethionine (RS) protein family with a C-terminal SPASM domain and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the PqqA peptide .

The significance of this ternary complex lies in several aspects:

First, it provides a molecular framework for substrate positioning. The tight binding of PqqA to PqqD (Kᴅ ~200 nM) likely positions the peptide in an optimal configuration for the subsequent radical-based chemistry catalyzed by PqqE .

Second, this arrangement may protect reactive radical intermediates from unwanted side reactions. The close proximity of the three components ensures that the radical species generated by PqqE's iron-sulfur cluster can be directed specifically to the target residues on PqqA .

Third, the formation of this complex might explain why biosynthetic intermediates in the PQQ pathway are not detected in culture supernatants of various PQQ-producing bacteria, as observed in studies with Acinetobacter calcoaceticus mutants . The tight complex formation would ensure that intermediates remain enzyme-bound throughout the pathway.

Bioinformatic analysis reveals that PqqD orthologues are associated with other RS-SPASM family proteins involved in peptide or protein modification . This suggests that the ternary complex formation observed in PQQ biosynthesis may represent a conserved mechanistic paradigm for radical SAM-mediated peptide modification pathways.

What are optimal protocols for recombinant expression and purification of PqqD from Acinetobacter species?

For successful recombinant expression and purification of PqqD from Acinetobacter species, the following optimized protocol can be implemented based on successful approaches with homologous proteins:

Expression System:

• Vector: pET24b or pET28a for C-terminal or N-terminal His₆-tag, respectively

• Host: E. coli BL21(DE3) or equivalent expression strain

• Induction: 0.5 mM IPTG when culture reaches OD₆₀₀ of 0.6-0.8

• Growth conditions: 18°C for 16-18 hours post-induction to maximize soluble protein yield

Purification Protocol for Native PqqD:

• Resuspend cells in 50 mM HEPES buffer (pH 6.8) at a 5:1 buffer:cell paste ratio

• Lyse cells by sonication and clarify lysate by centrifugation (20,000 rpm, 15 min)

• Load clarified lysate onto a pre-equilibrated 20-ml DEAE-Sepharose FF column

• Elute with a 0-0.5 M NaCl gradient in 50 mM HEPES buffer (pH 6.8) over 25 column volumes

  • PqqD typically elutes between 100-200 mM NaCl

• Pool PqqD-containing fractions and equilibrate with 17% saturated (NH₄)₂SO₄

• Load onto a pre-equilibrated HiPrep 16/10 Phenyl FF column

• Elute with a 17-0% (NH₄)₂SO₄ gradient over 15 column volumes

  • PqqD typically elutes between 9-12% saturated (NH₄)₂SO₄

• Concentrate pooled fractions and perform final purification by size exclusion chromatography using a 26/60 Sephacryl S-200 column equilibrated with 50 mM HEPES (pH 6.8) and 150 mM NaCl

For His₆-tagged PqqD:

• Use Ni-NTA affinity chromatography as the initial capture step

• Wash with buffer containing 20-30 mM imidazole to remove non-specifically bound proteins

• Elute with 250-300 mM imidazole

• Perform subsequent purification steps as needed

This protocol typically yields 6-8 mg of purified PqqD per liter of culture . Purified PqqD should be flash-frozen and stored at -80°C in small aliquots to maintain activity.

How can researchers accurately assess PqqD interactions with other proteins in the PQQ biosynthetic pathway?

Accurately assessing PqqD interactions with other proteins in the PQQ biosynthetic pathway requires a combination of complementary biophysical techniques and careful experimental design:

Binding Affinity Determination:

• Surface Plasmon Resonance (SPR):

  • Immobilize His₆-tagged PqqD on a Ni-NTA sensor chip

  • Flow potential binding partners (PqqA, PqqE) at various concentrations

  • For dual partner interactions, use sequential injections (e.g., PqqA followed by PqqE)

  • Calculate association and dissociation rate constants to determine Kᴅ values

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding thermodynamics without protein modification

  • Determines binding stoichiometry, enthalpy, and entropy changes

  • Requires careful buffer matching and concentration optimization

  • Particularly effective for high-affinity interactions (Kᴅ in nM range) like PqqD-PqqA

Complex Formation Analysis:

• Native Mass Spectrometry:

  • Preserves non-covalent interactions during ionization

  • Directly determines complex stoichiometry

  • Sample preparation critical: use volatile buffers like ammonium bicarbonate or ammonium acetate

  • Successfully captured the 1:1:1 MePqqA-D-E ternary complex

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS):

  • Determines absolute molecular weight of complexes

  • Distinguishes between different oligomeric states

  • Complements SAXS data for solution structure determination

• Analytical Ultracentrifugation (AUC):

  • Provides information on complex size, shape, and heterogeneity

  • Can detect multiple species in equilibrium

  • Particularly useful for characterizing dynamic complexes

For meaningful results, researchers should control protein quality (verify proper folding using circular dichroism), ensure consistent experimental conditions across techniques, and validate key findings with orthogonal methods. When discrepancies arise between techniques, consider protein dynamics or conformational changes that might affect binding properties in different experimental setups.

What approaches should be used to investigate potential intermediates in the PQQ biosynthetic pathway involving PqqD?

Investigating potential intermediates in the PQQ biosynthetic pathway involving PqqD presents significant challenges, as previous studies with Acinetobacter calcoaceticus, Methylobacterium organophilum, and Pseudomonas aureofaciens PQQ-deficient mutants failed to detect intermediates in culture supernatants, even under stress conditions . This suggests intermediates remain enzyme-bound throughout the pathway. To overcome these challenges, researchers should employ the following approaches:

In vitro Reconstitution System:

• Establish a minimal in vitro system with purified components (PqqA, PqqD, PqqE)

• Perform reactions with controlled stoichiometry under anaerobic conditions

• Use radical SAM reaction components: S-adenosylmethionine, sodium dithionite, and iron-sulfur cluster regeneration system

• Quench reactions at various time points for intermediate capture

Advanced Mass Spectrometry Approaches:

• Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS):

  • Use high-resolution instruments (Orbitrap or Q-TOF)

  • Develop multiple reaction monitoring (MRM) methods targeting predicted intermediate masses

  • Implement protein-centric top-down proteomics to detect modified PqqA peptides

• Chemical Crosslinking Mass Spectrometry:

  • Use bifunctional crosslinkers to "freeze" enzyme-intermediate complexes

  • Identify covalently trapped intermediates by MS/MS analysis

  • Map interaction surfaces between PqqD and modified PqqA intermediates

Spectroscopic Approaches:

• Time-resolved spectroscopy to capture transient intermediates:

  • UV-visible spectroscopy for chromophore development

  • Electron Paramagnetic Resonance (EPR) for radical intermediates

  • Resonance Raman spectroscopy for structural changes in the developing PQQ ring system

Genetic Approaches:

• Create conditional mutants in the PQQ biosynthetic genes

• Develop reporter systems linked to intermediate formation

• Use complementation assays with modified PqqD variants to identify critical residues

Computational Prediction:

• Use quantum mechanical calculations to predict intermediate structures

• Develop targeted analytical methods based on these predictions

• Perform molecular dynamics simulations of the PqqA-D-E complex to identify potential catalytic conformations

These multi-faceted approaches should be combined strategically, as no single method is likely to provide complete characterization of the pathway intermediates, especially given their apparent enzyme-bound nature throughout the biosynthetic process.

How can engineered PqqD fusion proteins be utilized in research applications?

Engineered PqqD fusion proteins represent valuable research tools for investigating PQQ biosynthesis and potentially developing biotechnological applications. Several successful PqqD fusion constructs have already been reported, providing precedent for future engineering efforts:

Existing Fusion Constructs:

• MePqqCD fusion: This naturally occurring fusion binds MePqqA with a Kᴅ of 180 ± 20 nM, similar to MePqqD alone (Kᴅ ~390 nM)

• MePqqDE fusion: An engineered fusion with a flexible (GGGGS)₄ linker that maintains MePqqA binding affinity (Kᴅ ~200 nM)

Research Applications:

• Streamlined Protein Production:

  • Expression of PqqD fusions can simplify purification of multi-component systems

  • Co-purification of interacting partners through affinity tags on PqqD

  • Enhanced stability of otherwise unstable PQQ pathway components

• Structural Studies:

  • Rigid fusion constructs for crystallography to capture interaction interfaces

  • SAXS analysis of fusion proteins to determine solution conformations of complexes

  • NMR studies using selective labeling strategies enabled by fusion constructs

• Functional Investigations:

  • Engineered PqqD fusions with fluorescent proteins for real-time monitoring of complex formation

  • FRET-based sensors to detect conformational changes during catalysis

  • PqqD fusions with proximity-dependent biotin ligases for capturing transient interactions

• Tool Development:

  • PqqD-based peptide delivery systems for targeting specific enzymes

  • Peptide display platforms utilizing PqqD's chaperone function

  • Biosensors based on PqqD-peptide recognition specificity

Experimental data confirms that PqqD maintains its peptide binding function in fusion constructs, as demonstrated by similar Kᴅ values across different constructs when binding MePqqA :

These consistent binding affinities suggest that PqqD's function is preserved in various fusion contexts, making it a versatile platform for protein engineering applications in PQQ biosynthesis research.

What insights does PqqD provide for understanding related peptide modification pathways involving radical SAM enzymes?

PqqD provides critical insights into a broader class of peptide modification pathways involving radical SAM enzymes, offering a potential paradigm for understanding related biosynthetic systems:

Conservation Patterns and Domain Architecture:
Bioinformatic analysis reveals that PqqD orthologues are specifically associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins . This conservation pattern suggests a common mechanistic framework for radical SAM-mediated peptide modifications.

Chaperone Function in Post-translational Modification:
PqqD's role as a peptide chaperone likely represents a conserved strategy for substrate recognition and positioning in radical SAM enzyme pathways. This function may be particularly important for pathways involving ribosomally synthesized and post-translationally modified peptides (RiPPs), where specific and precise modification of amino acid side chains is required .

Ternary Complex Formation:
The 1:1:1 ternary complex formation observed between PqqA, PqqD, and PqqE provides a structural model for similar interactions in other pathways. This arrangement likely serves multiple purposes:

• Ensuring substrate specificity

• Protecting reactive radical intermediates

• Facilitating sequential modifications by different enzymes

Evolutionary Implications:
The association of PqqD-like domains with diverse radical SAM enzymes across different biosynthetic pathways suggests:

• Modular evolution of post-translational modification systems

• Adaptation of a common protein-peptide recognition module for different substrates

• Conservation of mechanistic principles despite diverse end products

Structural Adaptations:
The conformational flexibility observed in PqqD structures (domain-swapped dimer versus compact monomer) might reflect adaptations to different peptide substrates and radical SAM enzyme partners across diverse pathways . This structural plasticity could be a key feature allowing PqqD-like domains to function across different biosynthetic systems.

These insights from PqqD studies have broader implications for understanding other peptide modification pathways, potentially guiding discovery and characterization of novel post-translational modifications mediated by radical SAM enzymes.

What are the remaining gaps in our understanding of PqqD function and structure?

Despite significant advances in PqqD research, several critical gaps remain in our understanding of its function and structure:

Structural Transition Mechanisms:
The discrepancy between the domain-swapped dimer observed in XcPqqD crystal structure and the monomeric globular form of KpPqqD in solution raises questions about potential functional conformational changes . It remains unclear whether PqqD undergoes structural transitions during its chaperone function and what triggers such changes. High-resolution structures of PqqD in complex with PqqA and PqqE would provide critical insights.

Peptide Recognition Specificity:
While PqqD binds PqqA with high affinity, the molecular determinants of this specificity remain poorly characterized. Key questions include:

• Which residues in PqqD form the peptide binding pocket?

• What sequence or structural features in peptide substrates are recognized by PqqD?

• How does PqqD distinguish between PqqA and other peptides?

Catalytic Contribution:
Beyond its chaperone function, PqqD might play additional roles in the enzymatic reaction:

• Does PqqD participate directly in catalysis or solely in substrate positioning?

• Does PqqD undergo conformational changes during the radical reaction?

• How does PqqD influence the reactivity of the radical SAM enzyme PqqE?

Species-Specific Adaptations:
Studies across different bacterial species (Acinetobacter, Klebsiella, Methylobacterium) show conservation of PqqD function, but potential adaptations to species-specific requirements remain unexplored . Comparative studies across diverse organisms would enhance our understanding of PqqD evolution.

Integration with Full Biosynthetic Pathway:
The relationship between the PqqA-D-E ternary complex and downstream enzymes (PqqB, PqqC) remains unclear:

• How are intermediates transferred between pathway components?

• Does PqqD participate in multiple steps of the pathway?

• What is the spatial and temporal organization of the complete biosynthetic machinery?

Dynamic Protein-Protein Interactions:
The kinetics and dynamics of complex assembly and disassembly are poorly understood:

• What is the order of binding events in ternary complex formation?

• How stable is the complex during catalysis?

• What triggers complex disassembly after the reaction?

Addressing these gaps will require integrated structural biology approaches, including hydrogen-deuterium exchange mass spectrometry, single-molecule techniques, and time-resolved spectroscopy, combined with genetic and biochemical studies across diverse bacterial systems.