Recombinant Bacillus licheniformis Molybdenum cofactor biosynthesis protein C (moaC)

What is Molybdenum Cofactor Biosynthesis Protein C (MoaC) and what is its role in biological systems?

Molybdenum cofactor (Moco) biosynthesis protein C (MoaC) is an essential enzyme involved in the early steps of molybdenum cofactor biosynthesis. Moco serves as a critical component for enzymes such as xanthine oxidase, sulfite oxidase, and nitrate reductase, playing a fundamental role in various biological systems . The molybdenum cofactor has a tricyclic pyranopterin structure with a dithiolene moiety that coordinates a molybdenum atom. This cofactor is synthesized through an evolutionarily conserved biosynthetic pathway found across archaea, eubacteria, and eukaryotes .

The importance of MoaC lies in its participation in the first step of Moco biosynthesis. In organisms like Escherichia coli, the Moco biosynthetic pathway involves at least seven proteins, with MoaC playing a crucial role in the initial conversion steps . Understanding the structure and function of MoaC from different organisms, including Bacillus licheniformis, provides valuable insights into the conservation and specialization of this essential metabolic pathway across species.

How does the structure of MoaC relate to its function in molybdenum cofactor biosynthesis?

The crystal structure studies of MoaC from various organisms reveal that it typically forms a hexamer with 32 symmetry. For instance, the MoaC from Sulfolobus tokodaii forms a hexamer arranged as a trimer of dimers with noncrystallographic 32 symmetry . Each MoaC monomer has a globular structure with an antiparallel β-sheet consisting of four β-strands and five α-helices located on the same side relative to the β-sheet . When two monomers form a dimer, their β-sheets connect to create a larger β-sheet structure.

The putative active site of MoaC is formed by highly conserved residues, which in S. tokodaii include Pro65–His68 (located in the loop between α3 and β2), Glu101–Glu103 (in α4), and from the opposing monomer of the dimer, Lys42 (in the loop between α1 and α2), Asp117 (in α4), and Lys120 (in α4) . These conserved residues create active sites located at each end of the dimer. This structural arrangement suggests a cooperative mechanism for substrate binding and catalysis, which is essential for the protein's role in Moco biosynthesis.

What methods are commonly used to express and purify recombinant Bacillus licheniformis MoaC?

Recombinant expression of B. licheniformis MoaC typically employs standard molecular cloning techniques. The gene encoding MoaC is amplified from B. licheniformis genomic DNA using PCR with specific primers containing appropriate restriction sites. The amplified gene is then cloned into an expression vector, commonly pET series vectors for E. coli expression systems, which contain a strong inducible promoter and an affinity tag (such as His-tag) for purification.

For protein expression, the recombinant plasmid is transformed into an E. coli expression strain like BL21(DE3). Expression is induced by adding IPTG when the culture reaches mid-log phase, typically at an OD600 of 0.6-0.8. The expression conditions, including temperature, IPTG concentration, and induction time, are optimized to maximize soluble protein yield.

Purification generally follows a multi-step approach:

• Affinity chromatography (if a His-tag is used, Ni-NTA resin)

• Ion-exchange chromatography to remove contaminating proteins

• Size-exclusion chromatography to obtain a homogeneous protein preparation and confirm the oligomeric state

The purity of the protein is assessed by SDS-PAGE, and its activity can be verified through enzyme assays measuring the conversion of appropriate substrates.

How does the structure of Bacillus licheniformis MoaC compare to MoaC proteins from other organisms?

For example, S. tokodaii MoaC contains an additional loop formed by the insertion of seven residues (Glu126, Asn127, Gly128, Gln129, Tyr130, Pro131, and Tyr132) between α5 and β4, which participates in intermonomer interactions . This additional loop forms hydrogen-bond interactions with the opposing subunit, potentially contributing to the stability of the dimer structure. Additionally, in S. tokodaii MoaC, three residues in the N-terminal loop region (Lys5, Ile6, and Val7) adopt a β-strand conformation (β0) and form hydrogen bonds with residues in adjacent dimers, creating an extended β-sheet consisting of ten β-strands from four monomers .

These structural differences likely reflect adaptations to different environmental conditions. B. licheniformis MoaC might exhibit similar structural adaptations, particularly considering its diverse ecological niches and probiotic properties.

What experimental approaches can be used to study the catalytic mechanism of B. licheniformis MoaC?

Several experimental approaches can be employed to elucidate the catalytic mechanism of B. licheniformis MoaC:

Site-directed mutagenesis: Based on sequence alignments with well-characterized MoaC proteins, conserved residues in the putative active site can be mutated to assess their roles in catalysis. For instance, mutations of residues corresponding to Pro65–His68, Glu101–Glu103, Lys42, Asp117, and Lys120 in S. tokodaii MoaC would provide insights into their functional importance .

Enzyme kinetics: Steady-state kinetic analyses with varying substrate concentrations can determine kinetic parameters (Km, kcat, kcat/Km) for wild-type and mutant proteins. These parameters would provide quantitative measures of the effects of mutations on substrate binding and catalysis.

Isothermal titration calorimetry (ITC): ITC can measure the thermodynamic parameters of substrate binding to MoaC, providing information about binding affinity, stoichiometry, and the enthalpy and entropy changes associated with binding.

X-ray crystallography: Determining the crystal structure of B. licheniformis MoaC in the presence of substrates, products, or substrate analogs would reveal the binding mode and potential conformational changes during catalysis. This approach was successful for S. tokodaii MoaC, which was crystallized and its structure determined at 2.2 Å resolution .

Mass spectrometry: Mass spectrometry can identify reaction intermediates and characterize post-translational modifications that might regulate MoaC activity.

How can crystallization conditions be optimized for structural studies of B. licheniformis MoaC?

Optimizing crystallization conditions for B. licheniformis MoaC requires a systematic approach:

• Protein preparation: Ensure high purity (>95%) and homogeneity of the protein sample. Verify the oligomeric state using size-exclusion chromatography, as MoaC typically forms hexamers .

• Initial screening: Employ commercial crystallization screens (e.g., Hampton Research, Molecular Dimensions) covering a wide range of conditions. Use techniques like sitting or hanging drop vapor diffusion to test multiple conditions simultaneously.

• Optimization strategies:

  • Fine-tune promising conditions by varying pH, precipitant concentration, and protein concentration

  • Test different additives and detergents that might promote crystal formation

  • Try seeding techniques to promote nucleation and crystal growth

  • Consider surface entropy reduction by mutating surface residues with high conformational entropy to alanine

  • Explore crystallization with substrates, products, or inhibitors to stabilize specific conformations

• Alternative approaches:

  • If full-length protein crystallization proves challenging, consider creating truncated constructs removing flexible regions

  • Co-crystallization with antibody fragments to provide additional crystal contacts

  • Use of fusion partners like T4 lysozyme that facilitate crystallization

For S. tokodaii MoaC, crystals were grown using the vapor diffusion method, and the structure was determined at 2.2 Å resolution . Similar approaches might be effective for B. licheniformis MoaC, potentially with adjustments to account for protein-specific properties.

What is the potential relationship between B. licheniformis MoaC and the organism's probiotic properties?

B. licheniformis is recognized as a probiotic organism with beneficial effects on both humans and animals. Studies have shown that B. licheniformis supplementation can improve growth performance, intestinal mucosal barrier functions, and immunity in weaned piglets . While the direct connection between MoaC and these probiotic properties is not explicitly established in the provided search results, several hypotheses can be proposed.

Molybdenum cofactors are essential for enzymes involved in nitrogen, carbon, and sulfur metabolism. By ensuring the proper functioning of these metabolic pathways through efficient Moco biosynthesis, MoaC might indirectly contribute to:

• Improved nutrient utilization: Enhanced activity of molybdoenzymes could lead to better nutrient processing, potentially explaining the improved growth performance observed in B. licheniformis-supplemented animals .

• Antioxidant defense: Moco-dependent enzymes like xanthine oxidase play roles in purine metabolism and redox reactions. B. licheniformis supplementation has been shown to increase activities of antioxidant enzymes like glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) while decreasing malondialdehyde (MDA) content .

• Intestinal health: B. licheniformis has been demonstrated to attenuate the negative effects of lipopolysaccharide (LPS) on intestinal morphology, particularly by reducing crypt depth and increasing villus height to crypt depth ratio in the jejunum . These effects could be partially mediated by metabolic pathways requiring Moco-dependent enzymes.

The following table summarizes the effects of B. licheniformis on serum antioxidant parameters in piglets:

These data suggest that B. licheniformis enhances antioxidant capacity and reduces oxidative damage , which might be indirectly influenced by MoaC's role in molybdoenzyme function.

What bioinformatic approaches can be used to predict the evolutionary conservation of the active site in B. licheniformis MoaC?

Several bioinformatic approaches can be employed to analyze the evolutionary conservation of B. licheniformis MoaC's active site:

Based on analysis of MoaC from other organisms, the putative active site residues that would likely be conserved in B. licheniformis MoaC include those corresponding to Pro65–His68, Glu101–Glu103, Lys42, Asp117, and Lys120 in S. tokodaii MoaC . These residues would be primary targets for conservation analysis and subsequent experimental validation.

How can isothermal titration calorimetry (ITC) be optimized for studying substrate binding to B. licheniformis MoaC?

Isothermal titration calorimetry (ITC) is a powerful technique for quantitatively measuring the thermodynamic parameters of biomolecular interactions. For studying substrate binding to B. licheniformis MoaC, the following optimization steps are recommended:

• Sample preparation:

  • Ensure high purity (>95%) of both protein and substrate

  • Dialyze protein and substrate in the exact same buffer to minimize heat of dilution effects

  • Determine optimal protein concentration (typically 10-50 μM) based on expected binding affinity

  • Use concentration of substrate in the syringe that is 10-20 times higher than the protein concentration

• Experimental parameters:

  • Optimize temperature (typically 25°C, but may vary depending on protein stability)

  • Set injection volume (2-10 μL) and spacing (180-300 seconds) to ensure return to baseline

  • Determine appropriate number of injections (20-30) to achieve saturation

  • Set appropriate reference power (5-10 μcal/sec) and stirring speed (300-400 rpm)

• Controls and validations:

  • Perform control experiments including buffer-into-buffer, substrate-into-buffer, and buffer-into-protein

  • Validate results with independent methods like fluorescence spectroscopy or surface plasmon resonance

  • Test different buffer conditions (pH, salt concentration) to optimize signal-to-noise ratio

• Data analysis:

  • Fit data using appropriate binding models (one-site, two-site, sequential binding)

  • Extract thermodynamic parameters: binding affinity (Kd), enthalpy change (ΔH), entropy change (ΔS), and stoichiometry (n)

  • Perform experiments at different temperatures to determine heat capacity changes (ΔCp)

This approach would provide comprehensive thermodynamic information about substrate binding to B. licheniformis MoaC, offering insights into the energetics and mechanisms of enzyme-substrate interactions.

What experimental design would be optimal for investigating the impact of specific mutations on B. licheniformis MoaC activity?

An optimal experimental design for investigating the impact of specific mutations on B. licheniformis MoaC activity would include the following components:

• Selection of mutation sites:

  • Target conserved residues in the putative active site based on sequence alignments with well-characterized MoaC proteins

  • Include residues corresponding to Pro65–His68, Glu101–Glu103, Lys42, Asp117, and Lys120 identified in S. tokodaii MoaC

  • Design additional mutations targeting residues involved in oligomerization, as MoaC functions as a hexamer

• Mutation strategy:

  • Perform alanine scanning mutagenesis for initial assessment

  • Follow up with more conservative substitutions to probe specific chemical properties

  • Create double or triple mutants to assess synergistic effects

  • Generate chimeric proteins with corresponding regions from other MoaC proteins to assess domain-specific functions

• Protein expression and purification:

  • Express wild-type and mutant proteins under identical conditions

  • Purify proteins using the same protocol to ensure comparability

  • Verify protein folding and oligomeric state using circular dichroism and size-exclusion chromatography

• Activity assays:

  • Develop a robust assay for MoaC activity, potentially using coupled enzyme systems or direct detection of product formation

  • Determine kinetic parameters (Km, kcat, kcat/Km) for all variants

  • Assess substrate specificity profiles for wild-type and mutant proteins

  • Test activity under various conditions (pH, temperature, salt concentration) to identify condition-dependent effects

• Structural characterization:

  • Determine crystal structures of key mutants to correlate activity changes with structural alterations

  • Perform molecular dynamics simulations to assess the impact of mutations on protein dynamics

  • Use hydrogen-deuterium exchange mass spectrometry to identify regions with altered conformational flexibility

• Statistical analysis:

  • Perform all experiments in triplicate to ensure reproducibility

  • Use appropriate statistical tests (t-test, ANOVA) to assess the significance of observed differences

  • Apply multiple comparison corrections when comparing numerous mutants

This comprehensive approach would provide mechanistic insights into how specific residues contribute to B. licheniformis MoaC function and could guide efforts to engineer the protein for enhanced activity or altered specificity.

How can two-dimensional NMR spectroscopy be used to study the dynamics of B. licheniformis MoaC?

Two-dimensional nuclear magnetic resonance (NMR) spectroscopy offers powerful tools for studying protein dynamics at atomic resolution. For B. licheniformis MoaC, the following NMR approaches would be valuable:

• Sample preparation:

  • Express isotopically labeled protein (15N, 13C, or both) in minimal media

  • For a protein the size of MoaC hexamer (~102 kDa), consider using deuteration to improve spectral quality

  • Optimize buffer conditions (pH, salt, temperature) for spectral quality while maintaining native structure

• NMR experiments for structural analysis:

  • 1H-15N HSQC to obtain protein "fingerprint" and assess folding

  • HNCA, HNCACB, CBCA(CO)NH for backbone assignments

  • 15N-NOESY-HSQC, 13C-NOESY-HSQC for structural constraints

  • Transverse relaxation optimized spectroscopy (TROSY) to improve spectral resolution for the large MoaC hexamer

• Dynamics measurements:

  • 15N relaxation measurements (T1, T2, heteronuclear NOE) to characterize ps-ns timescale motions

  • Relaxation dispersion experiments (CPMG, R1ρ) to probe μs-ms conformational exchange

  • ZZ-exchange spectroscopy to detect slower conformational transitions

• Ligand binding studies:

  • Chemical shift perturbation experiments to map binding interfaces

  • Transferred NOE experiments to determine bound ligand conformations

  • Saturation transfer difference (STD) NMR to identify ligand epitopes

• Data analysis and interpretation:

  • Model-free analysis of relaxation data to extract order parameters and correlation times

  • Map dynamics data onto the protein structure to correlate with functional regions

  • Compare dynamics in free and substrate-bound states to identify allosteric networks

Challenges for MoaC include its large size as a hexamer, which may necessitate studying smaller oligomeric states or domains. Alternatively, selective labeling strategies or specific probes at key residues could provide targeted information about dynamics at functionally important sites.

What are common challenges in expressing recombinant B. licheniformis MoaC and how can they be addressed?

Several challenges may arise when expressing recombinant B. licheniformis MoaC, along with potential solutions:

• Protein insolubility:

  • Challenge: Formation of inclusion bodies

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Reduce inducer concentration

    • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

    • Use solubility tags (SUMO, MBP, TrxA)

    • Optimize growth media and induction timing

• Incorrect oligomeric state:

  • Challenge: Failure to form the functional hexameric structure

  • Solutions:

    • Adjust buffer conditions (pH, salt concentration)

    • Add stabilizing agents (glycerol, specific metal ions)

    • Ensure proper disulfide bond formation if relevant

    • Consider co-expression with interaction partners

• Low expression levels:

  • Challenge: Poor yield of target protein

  • Solutions:

    • Optimize codon usage for expression host

    • Test different promoters and expression vectors

    • Screen various E. coli expression strains

    • Consider alternative expression systems (Bacillus, yeast)

• Protein instability:

  • Challenge: Rapid degradation of expressed protein

  • Solutions:

    • Add protease inhibitors during purification

    • Use expression strains deficient in specific proteases

    • Identify and modify protease-sensitive sites

    • Optimize storage conditions (buffer components, temperature)

• Post-translational modifications:

  • Challenge: Incorrect or missing modifications in the expression host

  • Solutions:

    • Express in hosts capable of performing relevant modifications

    • Engineer the protein to eliminate modification requirements

    • Perform in vitro modifications after purification

By systematically addressing these challenges, researchers can optimize the expression and purification of functional B. licheniformis MoaC for subsequent structural and functional studies.

How can conflicting data regarding the structure-function relationship of B. licheniformis MoaC be reconciled?

When faced with conflicting data about the structure-function relationship of B. licheniformis MoaC, researchers should adopt a systematic approach to reconciliation:

• Methodological considerations:

  • Evaluate the reliability and limitations of each experimental technique

  • Consider whether different methods might be probing different aspects of protein structure or function

  • Assess the experimental conditions (pH, temperature, buffer components) used in conflicting studies

  • Examine protein constructs for differences in tags, truncations, or mutations

• Structural heterogeneity:

  • Investigate whether MoaC might adopt multiple conformations with different functional properties

  • Consider allosteric regulation that might explain apparently contradictory results

  • Examine oligomerization states, as MoaC functions as a hexamer but might have different properties in other states

• Integration of multiple data types:

  • Combine structural data (X-ray crystallography, NMR, cryo-EM) with functional assays

  • Use computational methods (molecular dynamics, quantum mechanics/molecular mechanics) to bridge gaps between static structures and dynamic functions

  • Develop unified models that accommodate seemingly conflicting observations

• Targeted experiments to resolve conflicts:

  • Design experiments specifically addressing the contradictions

  • Use multiple orthogonal techniques to validate key findings

  • Consider time-resolved methods to capture transitional states

• Comparative analysis:

  • Compare with MoaC proteins from other organisms like S. tokodaii and E. coli to identify conserved features versus organism-specific adaptations

  • Examine whether conflicts might reflect species-specific differences in MoaC function

An example of structural differences that might lead to functional variations is seen between S. tokodaii and E. coli MoaC. S. tokodaii MoaC contains an additional loop formed by seven inserted residues that participate in intermonomer interactions, potentially contributing to the stability of the dimer structure . Such structural variations could explain functional differences observed between MoaC proteins from different organisms.

What are promising avenues for future research on B. licheniformis MoaC and its role in molybdenum cofactor biosynthesis?

Several promising research directions for B. licheniformis MoaC and its role in molybdenum cofactor biosynthesis include:

• Structural biology approaches:

  • Determine high-resolution crystal structures of B. licheniformis MoaC in different states (apo, substrate-bound, product-bound)

  • Use cryo-electron microscopy to visualize the hexameric assembly and potential conformational changes during catalysis

  • Apply hydrogen-deuterium exchange mass spectrometry to map dynamic regions involved in substrate binding and catalysis

• Systems biology integration:

  • Map the entire molybdenum cofactor biosynthesis pathway in B. licheniformis

  • Investigate regulatory mechanisms controlling MoaC expression and activity

  • Explore metabolic flux through the Moco biosynthesis pathway under different growth conditions

• Comparative studies:

  • Compare B. licheniformis MoaC with MoaC proteins from other probiotics to identify unique features

  • Investigate how MoaC has evolved across diverse bacteria, particularly those adapted to different ecological niches

  • Examine the relationship between MoaC variations and the metabolic versatility of different bacterial species

• Connection to probiotic properties:

  • Investigate how MoaC-dependent molybdoenzymes contribute to the beneficial effects of B. licheniformis in the host gut

  • Study whether MoaC activity correlates with antioxidant capacity and other probiotic benefits observed in B. licheniformis supplementation

  • Develop B. licheniformis strains with modified MoaC to enhance specific probiotic properties

• Technological applications:

  • Explore the potential use of B. licheniformis MoaC for in vitro synthesis of molybdenum cofactors

  • Investigate applications in bioremediation, as molybdoenzymes often participate in the transformation of environmental pollutants

  • Develop biosensors based on molybdoenzymes dependent on MoaC activity

These research directions would advance our understanding of molybdenum cofactor biosynthesis in B. licheniformis and potentially reveal connections to its probiotic properties, opening new avenues for applications in both basic science and biotechnology.