Recombinant Dichelobacter nodosus Phosphoserine aminotransferase (serC)

What is Phosphoserine aminotransferase (serC) in Dichelobacter nodosus and what is its biochemical function?

Phosphoserine aminotransferase (serC) in Dichelobacter nodosus is an enzyme (EC 2.6.1.52) involved in serine biosynthesis. This enzyme catalyzes the conversion of 3-phosphohydroxypyruvate to L-phosphoserine, an essential step in the serine biosynthetic pathway. The full-length protein consists of 358 amino acids with a well-conserved sequence across bacterial species . Functionally, serC plays a crucial role in amino acid metabolism, particularly in organisms like D. nodosus, where metabolic adaptations are critical for survival in host environments. The enzyme requires pyridoxal-5'-phosphate (PLP) as a cofactor to facilitate the transamination reaction. SerC's activity is particularly important in bacterial survival under stress conditions, as evidenced by homologous proteins in mycobacteria that respond to serine stress .

How can I verify the identity and purity of recombinant Dichelobacter nodosus serC?

Verification of identity and purity requires multiple complementary techniques:

• SDS-PAGE analysis: The recombinant serC protein should appear as a single band at approximately 39 kDa (full-length protein). Purity levels above 85% are typically considered acceptable for research applications .

• Western blotting: For tagged versions (e.g., His-tagged), use tag-specific antibodies such as Ni-NTA HRP conjugate. This approach successfully identified a Trx-6xHis-tagged recombinant protein from D. nodosus in previous studies .

• Mass spectrometry:

  • Peptide mass fingerprinting following tryptic digestion

  • LC-MS/MS for sequence verification against the expected amino acid sequence

• Enzymatic activity assay: Measure the conversion of 3-phosphohydroxypyruvate to L-phosphoserine using coupled enzyme assays or direct product detection.

• Circular dichroism: Verify proper folding by analyzing secondary structure content.

What expression systems are optimal for producing recombinant D. nodosus serC protein?

Based on available data and general recombinant protein methodology, several expression systems can be considered:

The selection of expression vector should include appropriate tags for purification (e.g., His-tag) and fusion partners that enhance solubility (e.g., Thioredoxin, SUMO, or MBP).

What challenges might arise during expression of recombinant D. nodosus serC and how can they be addressed?

Several challenges may arise when expressing D. nodosus proteins, based on research with other proteins from this organism:

• Host toxicity: Similar to observations with D. nodosus fimbrial proteins , serC might exhibit toxicity to host cells.

  • Solution: Use tightly regulated expression systems, reduce temperature, add glucose to suppress basal expression, and employ expression hosts designed for toxic proteins (e.g., C41/C43 E. coli strains).

• Protein solubility: Aminotransferases can form inclusion bodies.

  • Solution: Express as fusion proteins with solubility enhancers (Thioredoxin, SUMO, MBP), optimize buffer conditions, or develop refolding protocols.

• Protein stability: SerC may exhibit limited stability.

  • Solution: Include stabilizing agents (glycerol 20-50%, reducing agents) in storage buffers, aliquot and avoid freeze-thaw cycles .

• Limited activity: Recombinant enzymes may show suboptimal activity.

  • Solution: Ensure presence of PLP cofactor, optimize buffer composition, and verify proper oligomeric state.

• Purification challenges: Multiple steps may be needed for high purity.

  • Solution: Implement multi-step purification strategies combining affinity chromatography with size-exclusion and/or ion-exchange methods.

What is the recommended protocol for purification of recombinant D. nodosus serC protein?

Based on established methods for similar proteins:

Step 1: Initial lysis and clarification

• Resuspend cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 5 mM β-mercaptoethanol)

• Lyse cells via sonication or pressure homogenization

• Centrifuge at 15,000×g for 30 minutes at 4°C to remove debris

Step 2: Affinity chromatography (for His-tagged protein)

• Apply clarified lysate to Ni-NTA column pre-equilibrated with binding buffer

• Wash with 20-30 column volumes of wash buffer (lysis buffer with 20-30 mM imidazole)

• Elute protein with elution buffer (lysis buffer with 250-300 mM imidazole)

• Monitor purification by SDS-PAGE analysis

Step 3: Secondary purification

• Perform size-exclusion chromatography using Superdex 200 column in buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, and 10% glycerol

• Pool fractions containing pure protein

Step 4: Storage

• Add glycerol to a final concentration of 20-50%

• Aliquot and store at -20°C or -80°C to avoid repeated freeze-thaw cycles

How can I assess the enzymatic activity of recombinant D. nodosus serC?

Enzymatic activity of serC can be measured using several complementary approaches:

• Coupled enzyme assay:

  • The phosphoserine produced by serC is converted to serine by phosphoserine phosphatase

  • Serine is then quantified via reaction with periodate and measurement of formaldehyde production

  • Alternatively, use NAD(P)H-dependent coupled assays and monitor absorbance at 340 nm

• Direct product detection:

  • HPLC separation of substrate and product

  • LC-MS/MS for sensitive detection and quantification

  • Colorimetric assays for phosphate release

• Standard reaction conditions:

  • Buffer: 50 mM HEPES pH 7.5, 5 mM MgCl₂, 1 mM DTT

  • Substrates: 3-phosphohydroxypyruvate (0.1-1 mM), glutamate (1-10 mM)

  • Cofactor: PLP (0.1 mM)

  • Temperature: 37°C

  • Time: 10-30 minutes

• Kinetic parameters determination:

  • Vary substrate concentration to determine K_m, V_max, and k_cat

  • Analyze data using Michaelis-Menten or Lineweaver-Burk plots

What is the role of serC in D. nodosus virulence and pathogenicity?

While direct evidence for serC's role in D. nodosus virulence is limited in the provided search results, insights can be drawn from related research:

• Metabolic adaptation: SerC's role in serine biosynthesis likely contributes to bacterial survival in nutrient-limited environments. Studies in mycobacteria have demonstrated that serC responds to environmental stress and influences biofilm formation .

• Stress response: In mycobacteria, NapR regulates serC expression in response to serine stress . Similar regulatory mechanisms may exist in D. nodosus, allowing adaptation to host environments.

• Biofilm formation: SerC has been implicated in biofilm formation in mycobacteria . If similar functions exist in D. nodosus, this could contribute to virulence, as biofilms enhance resistance to antimicrobials and host defense mechanisms.

• Potential vaccine target: The conservation of serC across bacterial species and its essential metabolic function make it a potential target for vaccine development, similar to approaches using other D. nodosus proteins .

Further research is needed to directly establish serC's role in D. nodosus virulence, potentially through knockout studies or expression analysis during infection.

How does serC interact with other proteins in D. nodosus metabolic pathways?

SerC functions within an interconnected metabolic network:

• Serine biosynthesis pathway interactions:

  • SerC receives its substrate from SerA (phosphoglycerate dehydrogenase)

  • SerC product is utilized by SerB (phosphoserine phosphatase)

  • This three-enzyme pathway represents a critical link between glycolysis and amino acid metabolism

• Regulatory interactions:

  • By analogy with other bacterial systems, serC may be regulated by transcription factors similar to NapR found in mycobacteria

  • Potential protein-protein interactions with regulatory elements that sense amino acid availability

• Metabolic network integration:

  • Links to folate metabolism (serine is required for single-carbon transfer reactions)

  • Connections to cysteine biosynthesis (serine serves as precursor)

  • Integration with central carbon metabolism through 3-phosphoglycerate

A predictive model of serC interactions can be constructed based on known bacterial metabolic networks, though experimental verification through techniques like pull-down assays, bacterial two-hybrid systems, or co-immunoprecipitation would be necessary for D. nodosus-specific interactions.

How do mutations in serC affect enzyme function and D. nodosus fitness?

Mutation analysis of serC can provide insights into structure-function relationships and bacterial fitness:

• Catalytic site mutations:

  • Mutations in PLP-binding residues typically abolish activity

  • Substrate binding site mutations may alter enzyme kinetics (K_m, k_cat)

  • Stability-enhancing mutations might increase enzyme longevity

• Fitness impact:

  • Complete loss of serC function would likely result in serine auxotrophy

  • Reduced activity might compromise growth in serine-limited environments

  • Hyperactive mutations could potentially disrupt amino acid homeostasis

• Experimental approaches:

  • Site-directed mutagenesis of conserved residues

  • Random mutagenesis coupled with selection/screening

  • Expression of mutant variants in serC-deficient strains to assess complementation

• Structural implications:

  • Mutations affecting dimerization interfaces may alter quaternary structure

  • Surface mutations might impact protein-protein interactions

  • Changes in flexible loops could alter substrate access or product release

Understanding these relationships requires integrated approaches combining biochemical characterization with in vivo fitness studies.

What advanced analytical techniques can be applied to study serC structure-function relationships?

Several sophisticated techniques can provide deeper insights into serC structure and function:

• X-ray crystallography and cryo-EM:

  • Determination of three-dimensional structure at atomic resolution

  • Co-crystallization with substrates, products, or inhibitors to visualize binding modes

  • Structural comparison with homologous enzymes

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Probes protein dynamics and conformational changes

  • Identifies regions with differential solvent accessibility upon ligand binding

  • Maps protein-protein interaction interfaces

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Studies protein dynamics in solution

  • Maps chemical shift perturbations upon ligand binding

  • Determines binding constants and exchange rates

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to monitor conformational changes

  • Optical tweezers to study enzyme mechanics

  • Single-molecule enzymology to detect reaction intermediates

• Computational approaches:

  • Molecular dynamics simulations to study protein flexibility

  • Quantum mechanics/molecular mechanics (QM/MM) to model the catalytic mechanism

  • In silico docking and virtual screening for inhibitor discovery

These techniques, combined with biochemical and genetic approaches, provide a comprehensive understanding of serC function.

How can recombinant D. nodosus serC be utilized in vaccine development strategies?

Recombinant D. nodosus serC presents several opportunities for vaccine development:

• Subunit vaccine approaches:

  • Purified recombinant serC can be formulated with appropriate adjuvants

  • Proteins can be delivered alone or as part of multi-antigen formulations

  • Expression systems like yeast might provide advantages for large-scale production

• Epitope identification strategies:

  • Computational prediction of B-cell and T-cell epitopes within serC sequence

  • Experimental validation through epitope mapping techniques

  • Development of epitope-based synthetic peptide vaccines

• Vector-based delivery systems:

  • Expression of serC in attenuated bacterial or viral vectors

  • DNA vaccines encoding serC

  • mRNA vaccine approaches

• Potential advantages of serC as a vaccine target:

  • Conservation across strains (potentially broader protection)

  • Essential metabolic function (reduced risk of escape mutants)

  • Recombinant production established and scalable

• Evaluation protocol:

  • In vitro neutralization assays

  • Animal immunization studies with challenge

  • Analysis of humoral and cell-mediated immune responses

Similar approaches have been considered for other D. nodosus proteins, such as fimbrial subunits, which have been expressed as recombinant proteins for potential vaccine development .

What research gaps exist in our understanding of D. nodosus serC and how might they be addressed?

Several significant knowledge gaps remain regarding D. nodosus serC:

• Regulatory mechanisms:

  • Limited understanding of how serC expression is regulated in D. nodosus

  • Potential research approach: Transcriptomic analysis under various conditions, promoter mapping, identification of regulatory proteins (similar to NapR in mycobacteria)

• Host-pathogen interactions:

  • Unknown whether serC is expressed during infection or contributes to virulence

  • Research strategy: In vivo expression analysis during infection, serC knockout studies

• Structural information:

  • No crystal structure available for D. nodosus serC

  • Approach: Protein crystallization trials, structural determination by X-ray crystallography or cryo-EM

• Species-specific properties:

  • Limited characterization of biochemical properties specific to D. nodosus serC

  • Strategy: Comparative enzymology with homologs from other species, substrate specificity profiling

• Inhibitor development:

  • No specific inhibitors reported for D. nodosus serC

  • Approach: High-throughput screening, structure-based design, fragment-based drug discovery

These gaps present opportunities for researchers to make significant contributions to the field, potentially leading to novel therapeutic or preventive strategies against D. nodosus infections.

What protocols should be followed for long-term storage and handling of recombinant D. nodosus serC?

Optimal storage and handling procedures for maintaining serC stability and activity:

• Short-term storage (up to 1 week):

  • Store at 4°C in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT

  • Avoid repeated freeze-thaw cycles

• Long-term storage:

  • Add glycerol to a final concentration of 20-50%

  • Aliquot in small volumes to avoid freeze-thaw cycles

  • Store at -20°C for up to 6 months or -80°C for up to 12 months

• Reconstitution of lyophilized protein:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration for storage

  • Aliquot for long-term storage at -20°C/-80°C

• Handling precautions:

  • Briefly centrifuge vials before opening to bring contents to the bottom

  • Maintain enzyme in reducing conditions (1-5 mM DTT or β-mercaptoethanol)

  • Consider adding PLP (0.1 mM) to stabilize the enzyme

• Quality control:

  • Periodically verify activity using standardized assays

  • Check protein integrity by SDS-PAGE after extended storage

Following these guidelines maximizes protein stability and functional integrity for research applications.

How can I troubleshoot common issues with recombinant D. nodosus serC experiments?

For enzyme activity issues, a systematic analysis of reaction conditions (pH, temperature, cofactors, substrates) can help identify optimal parameters for D. nodosus serC function.