Recombinant Dichelobacter nodosus Serine hydroxymethyltransferase (glyA)

What is Serine hydroxymethyltransferase (glyA) and what is its biochemical function in Dichelobacter nodosus?

Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) in Dichelobacter nodosus is a crucial enzyme that catalyzes the reversible conversion of serine to glycine with concurrent transfer of a one-carbon unit to tetrahydrofolate, forming 5,10-methylenetetrahydrofolate. This reaction is central to one-carbon metabolism, which is essential for nucleotide synthesis, amino acid metabolism, and methylation reactions. In D. nodosus (strain VCS1703A), this enzyme is encoded by the glyA gene and consists of 417 amino acids as indicated by the expression region . The enzyme plays a pivotal role in bacterial metabolism, particularly in providing one-carbon units for purine and thymidylate synthesis, which are crucial for DNA replication and cell division.

How does the structure of D. nodosus SHMT compare to SHMT from other bacterial species?

D. nodosus SHMT shares the conserved catalytic core structure typical of bacterial SHMTs, including specific binding domains for pyridoxal 5′-phosphate (PLP), its essential cofactor. The protein sequence (UniProt A5EVR7) reveals conserved residues for PLP binding and catalysis. Based on sequence analysis, D. nodosus SHMT contains characteristic motifs including the GFAAYSQ region that is part of the PLP-binding site, which is highly conserved across bacterial SHMTs. The protein sequence also indicates the presence of key structural elements including:

• N-terminal domain (residues 1-90): Involved in tetramer formation

• Central catalytic domain (residues 91-313): Contains the PLP-binding site

• C-terminal domain (residues 314-417): Contributes to substrate specificity

Unlike some mammalian SHMTs that can localize to different cellular compartments, bacterial SHMTs including D. nodosus are cytosolic enzymes that typically function as homotetramers.

What purification methods are most effective for isolating active recombinant D. nodosus SHMT?

For successful purification of recombinant D. nodosus SHMT with retained activity, a multi-stage approach is recommended:

• Initial Clarification: After expression in mammalian cells (as specified in the datasheet), perform cell lysis under gentle conditions (e.g., sonication with pulse intervals or enzymatic lysis) to prevent protein denaturation .

• Chromatographic Separation:

  • IMAC (Immobilized Metal Affinity Chromatography): If the recombinant protein includes a histidine tag

  • Ion Exchange Chromatography: Based on the theoretical pI of the protein

  • Size Exclusion Chromatography: For final polishing and buffer exchange

• Activity Preservation Measures:

  • Include PLP (5-20 μM) in all purification buffers

  • Maintain reducing conditions with 1-5 mM DTT or β-mercaptoethanol

  • Use buffer systems in the pH range 7.0-8.0 to maintain stability

  • Work at 4°C throughout the purification process

The purity goal should be >85% as assessed by SDS-PAGE, consistent with the specifications noted in the product datasheet .

How should researchers design enzyme activity assays for D. nodosus SHMT?

When designing enzyme activity assays for D. nodosus SHMT, researchers should consider multiple approaches based on the reaction catalyzed:

Spectrophotometric Coupled Assays:

• Measure the formation of 5,10-methylenetetrahydrofolate by coupling with NADPH-dependent methylenetetrahydrofolate reductase

• Monitor absorbance changes at 340 nm due to NADPH oxidation

• Use the following reaction conditions:

  • 50 mM phosphate buffer (pH 7.4)

  • 1-2 mM serine

  • 0.4 mM tetrahydrofolate

  • 50 μM PLP

  • 0.1-0.5 μM recombinant D. nodosus SHMT

  • Temperature: 25°C (standard) or 37°C (physiological)

Radiometric Assays:

• Use 14C-labeled serine as substrate

• Quantify the formation of [14C]glycine after separation by HPLC or TLC

• This approach offers higher sensitivity for kinetic studies

Data Analysis:

• Calculate initial velocities from the linear portion of progress curves

• Determine kinetic parameters (Km, kcat, Vmax) using appropriate software (GraphPad Prism, SigmaPlot, etc.)

• Use Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee plots for data visualization

What strategies can be employed to study the substrate specificity of D. nodosus SHMT?

Comprehensive investigation of D. nodosus SHMT substrate specificity requires multiple experimental approaches:

Alternative Substrate Screening:

• Test structurally related amino acids (e.g., D-serine, threonine, alanine) at 1-5 mM concentrations

• Examine various folate derivatives as potential one-carbon acceptors

• Measure relative activity using standard assay conditions

Structural Biology Approaches:

• Crystallography with various substrates or substrate analogs bound

• Molecular docking simulations to predict binding modes

• Hydrogen-deuterium exchange mass spectrometry to identify substrate interaction sites

Site-Directed Mutagenesis Studies:

• Identify conserved residues in the active site based on sequence alignment

• Generate point mutations of these residues

• Characterize mutant enzymes for altered substrate preference

• Measure kinetic parameters for each mutant with various substrates

Competition Assays:

• Determine IC50 values for substrate analogs

• Calculate Ki values using appropriate inhibition models

• Compare binding affinity patterns across different potential substrates

When reporting substrate specificity data, include the following parameters for each substrate:

What are the optimal conditions for reconstitution and storage of recombinant D. nodosus SHMT?

According to the product datasheet, specific guidelines should be followed for optimal reconstitution and storage of recombinant D. nodosus SHMT:

Reconstitution Protocol:

• Centrifuge the vial briefly to collect contents at the bottom before opening

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

• Add glycerol to a final concentration of 5-50% (the default recommendation is 50%)

• Mix gently to avoid protein denaturation

• Allow the protein to stabilize at room temperature for 15-30 minutes

Storage Recommendations:

• Prepare working aliquots to avoid repeated freeze-thaw cycles

• For extended storage: -20°C or -80°C (with 50% glycerol)

• For short-term storage (up to one week): 4°C

• The shelf life in liquid form is approximately 6 months at -20°C/-80°C

• The shelf life in lyophilized form is approximately 12 months at -20°C/-80°C

Critical Factors Affecting Stability:

• Avoid repeated freezing and thawing cycles

• Include stabilizing agents during storage:

  • 50% glycerol (cryoprotectant)

  • 1-5 mM DTT (prevents oxidation of cysteine residues)

  • 20-50 μM PLP (maintains cofactor saturation)

• Store in small aliquots (50-100 μL) to minimize freeze-thaw cycles

• Use low-binding microcentrifuge tubes to prevent protein adsorption

How can researchers investigate the role of D. nodosus SHMT in bacterial pathogenesis?

Investigating the role of D. nodosus SHMT in pathogenesis requires a multi-faceted approach:

Genetic Manipulation Studies:

• Create glyA knockout or knockdown strains

• Develop conditional expression systems to control glyA expression

• Complement mutants with wild-type or modified glyA genes

• Assess virulence phenotypes in appropriate infection models

Comparative Genomics and Transcriptomics:

• Analyze glyA expression patterns during infection stages

• Compare expression levels between virulent and avirulent strains

• Identify potential regulatory elements controlling glyA expression

• Examine changes in one-carbon metabolism during host infection

Biochemical Approaches:

• Compare enzymatic properties of SHMT from virulent vs. avirulent strains

• Identify post-translational modifications that occur during infection

• Investigate potential moonlighting functions beyond canonical SHMT activity

• Examine interactions with host proteins or metabolites

Inhibition Studies:

• Test selective SHMT inhibitors in infection models

• Evaluate metabolic rescue experiments with glycine and nucleotide precursors

• Assess synergy between SHMT inhibition and conventional antimicrobials

• Develop structure-based design of inhibitors targeting unique features of the D. nodosus enzyme

Data Interpretation Framework:

• Correlate enzymatic activity with virulence measurements

• Distinguish direct from indirect effects through complementation studies

• Consider metabolic network adaptations that may compensate for SHMT inhibition

• Integrate findings with broader understanding of D. nodosus pathogenesis in foot rot disease

What experimental designs are suitable for studying potential inhibitors of D. nodosus SHMT?

Developing a comprehensive inhibitor screening and characterization pipeline for D. nodosus SHMT includes:

Primary Screening Approaches:

• High-throughput spectrophotometric assays

  • Measure SHMT activity in 96 or 384-well format

  • Screen compound libraries at single concentrations (typically 10-50 μM)

  • Include appropriate controls (known inhibitors, vehicle controls)

• Fragment-based screening

  • Use thermal shift assays to identify stabilizing fragments

  • Employ NMR-based screening for fragment binding

• Virtual screening

  • Structure-based docking of compound libraries

  • Pharmacophore modeling based on substrate interactions

Secondary Validation and Characterization:

• Dose-response analysis

  • Determine IC50 values for hit compounds

  • Establish complete inhibition curves

• Mechanism of action studies

  • Determine inhibition type (competitive, noncompetitive, uncompetitive)

  • Measure Ki values using appropriate kinetic models

• Selectivity profiling

  • Test activity against human SHMT isoforms

  • Evaluate effects on related PLP-dependent enzymes

• Structure-activity relationship analysis

  • Test structural analogs of lead compounds

  • Identify chemical moieties critical for inhibition

Advanced Characterization:

• Binding studies

  • Isothermal titration calorimetry for thermodynamic parameters

  • Surface plasmon resonance for binding kinetics

• Co-crystallization with inhibitors

  • Determine binding modes and key interactions

  • Inform structure-based optimization

• Cellular activity evaluation

  • Measure effects on D. nodosus growth

  • Assess impact on one-carbon metabolism in vivo

How can site-directed mutagenesis be employed to investigate the catalytic mechanism of D. nodosus SHMT?

Site-directed mutagenesis provides powerful insights into SHMT's catalytic mechanism. A systematic approach should include:

Target Residue Selection Strategy:

• Conserved active site residues based on sequence alignment with well-characterized SHMTs

• PLP-binding site residues (typically includes a catalytic lysine forming Schiff base with PLP)

• Substrate binding pocket residues

• Residues involved in quaternary structure formation

Recommended Mutation Types:

• Conservative mutations (e.g., Lys→Arg, Asp→Glu) to probe charge requirements

• Non-conservative mutations to drastically alter chemical properties

• Alanine-scanning mutagenesis to identify essential side chains

• Introduction of unnatural amino acids for specialized mechanistic studies

Functional Analysis of Mutants:

• Expression and purification under identical conditions to wild-type

• Spectroscopic characterization:

  • UV-visible spectroscopy to assess PLP binding

  • Circular dichroism to confirm structural integrity

  • Fluorescence spectroscopy to examine conformational changes

• Kinetic characterization:

  • Determine Km and kcat values for all substrates

  • Measure effects on reaction specificity

  • Analyze pH and temperature dependence profiles

• Thermodynamic stability assessment:

  • Thermal denaturation studies

  • Chemical denaturation profiles

Data Interpretation Framework:

• Correlate structural locations with functional impacts

• Compare effects across different substrates to identify substrate-specific interactions

• Develop refined catalytic mechanism models based on mutational effects

• Integrate findings with structural data when available

Key residues that typically warrant investigation in bacterial SHMTs include:

• The PLP-binding lysine residue (forming internal aldimine)

• Residues coordinating the phosphate group of PLP

• Basic residues interacting with the carboxylate of serine

• Residues forming the THF binding pocket

How can researchers address problems with low activity or inactivity of recombinant D. nodosus SHMT?

When facing low activity or inactivity issues with recombinant D. nodosus SHMT, researchers should systematically troubleshoot using this decision tree approach:

Cofactor-Related Issues:

• Ensure PLP saturation by:

  • Adding fresh PLP (50-100 μM) to enzyme preparations

  • Incubating the enzyme with excess PLP (100 μM) followed by desalting

  • Checking for characteristic PLP absorption peak (425-435 nm)

• Verify PLP quality:

  • Use freshly prepared PLP solutions protected from light

  • Check PLP purity by spectroscopic analysis

Protein Integrity Problems:

• Assess protein folding:

  • Perform circular dichroism spectroscopy

  • Use fluorescence spectroscopy to examine tertiary structure

  • Consider native PAGE to check quaternary structure

• Verify protein integrity:

  • Confirm correct molecular weight by mass spectrometry

  • Check for proteolytic degradation by SDS-PAGE

  • Add protease inhibitors during purification and storage

Buffer and Reaction Condition Optimization:

• Test different buffer systems:

  • Phosphate buffer (pH 7.0-8.0)

  • HEPES or Tris buffer (pH 7.2-8.0)

  • Include 50-150 mM NaCl for stability

• Optimize reaction conditions:

  • Test activity across pH range (6.5-8.5)

  • Vary temperature (25-42°C)

  • Add stabilizing agents (glycerol, BSA, reducing agents)

Expression and Purification Factors:

• Modify expression conditions:

  • Try different expression systems (mammalian, bacterial, insect cells)

  • Reduce expression temperature to enhance proper folding

  • Include chaperones during expression

• Adjust purification strategy:

  • Use milder elution conditions

  • Include PLP in all purification buffers

  • Minimize exposure to extreme pH or salt conditions

A systematic examination of these factors, preferably in a controlled experimental design, will help identify and resolve the source of activity problems.

How can researchers interpret contradictory results in D. nodosus SHMT inhibition studies?

When facing contradictory results in inhibition studies, apply this systematic approach to reconcile discrepancies:

Inhibitor-Related Factors:

• Evaluate compound properties:

  • Solubility limitations in assay buffers

  • Intrinsic fluorescence or absorbance that interferes with assay readout

  • Aggregation potential at tested concentrations

• Consider compound quality:

  • Purity differences between studies

  • Stereochemical variations or racemization

  • Stability under assay conditions

Data Analysis Framework:

• Create a comprehensive comparison table with standardized parameters:

• Perform cross-validation experiments:

  • Reproduce published methods side-by-side

  • Test inhibitors under identical conditions

  • Consider blind testing by independent researchers

• Mechanistic reconciliation:

  • Different inhibition mechanisms at different concentrations

  • Allosteric effects vs. active site competition

  • Time-dependent inhibition phenomena

By systematically addressing these factors, researchers can develop a unified understanding of seemingly contradictory inhibition data and establish more robust inhibition models.

What strategies can address protein aggregation or precipitation issues with D. nodosus SHMT?

Protein aggregation or precipitation of recombinant D. nodosus SHMT can significantly impact experimental outcomes. Here's a comprehensive troubleshooting approach:

Prevention Strategies During Handling:

• Buffer optimization:

  • Test buffer screening with varying pH (6.8-8.2)

  • Evaluate different ionic strengths (50-300 mM NaCl)

  • Include stabilizing additives:

    • 5-10% glycerol

    • 0.1-0.5 M trehalose or sucrose

    • 0.1-1.0 mg/mL BSA as a carrier protein

• Physical handling considerations:

  • Avoid rapid temperature changes

  • Prevent air-liquid interface exposure (minimize vortexing)

  • Use low-protein-binding tubes and pipette tips

  • Filter solutions through 0.22 μm membranes before use

Resolving Existing Aggregation Issues:

• Gentle disaggregation approaches:

  • Centrifuge at 15,000 × g for 10 minutes to remove large aggregates

  • Filter through 0.22 μm filters for analytic applications

  • Try mild sonication (low power, ice bath, short pulses)

• Chemical chaperone addition:

  • L-arginine (50-100 mM)

  • Non-detergent sulfobetaines (5-10 mM)

  • Low concentrations of mild detergents (0.01-0.05% Tween-20)

Analytical Tools for Monitoring Aggregation:

• Dynamic light scattering for particle size distribution

• Size exclusion chromatography to quantify monomer/oligomer/aggregate ratios

• Analytical ultracentrifugation for detailed solution behavior

• Thioflavin T binding to detect amyloid-like aggregation

Root Cause Analysis Matrix:

Implementing these strategies will help maintain D. nodosus SHMT in a soluble, active state suitable for downstream applications.

How can researchers leverage D. nodosus SHMT for comparative studies across bacterial species?

Comparative studies of SHMT across bacterial species offer valuable insights into evolution, adaptation, and potential antimicrobial targets. A comprehensive approach should include:

Sequence and Structure-Based Comparisons:

• Multi-sequence alignment analysis:

  • Compare D. nodosus SHMT sequence with orthologs from related and distant bacteria

  • Identify conserved residues versus lineage-specific variations

  • Construct phylogenetic trees to trace evolutionary relationships

• Structural comparison:

  • Analyze available crystal structures or create homology models

  • Identify structural differences in active sites, oligomerization interfaces, and allosteric sites

  • Map sequence variations onto structural models to identify functionally relevant differences

Functional Comparison Framework:

• Enzyme kinetics standardization:

  • Express and purify multiple bacterial SHMTs under identical conditions

  • Compare kinetic parameters under standardized assay conditions

  • Develop a comprehensive kinetic profile for each ortholog

• Inhibition profile comparison:

  • Test inhibitor panels across multiple bacterial SHMTs

  • Identify species-specific inhibition patterns

  • Quantify selectivity indices for potential antimicrobial development

• Determine species-specific characteristics:

  • pH and temperature optima

  • Substrate specificity profiles

  • Cofactor requirements and binding affinities

  • Quaternary structure stability

Experimental Design Considerations:

• Select species representing diverse phylogenetic groups

• Include both pathogenic and non-pathogenic species

• Consider extremophiles to explore adaptation to environmental niches

• Include human SHMT isoforms as references for selectivity studies

This comprehensive approach will yield insights into SHMT's evolutionary adaptations and identify species-specific features that can be exploited for selective inhibition.

What are the most promising approaches for studying the potential of D. nodosus SHMT as an antimicrobial target?

Evaluating D. nodosus SHMT as an antimicrobial target requires a systematic, multi-disciplinary approach:

Target Validation Studies:

• Genetic essentiality assessment:

  • Conditional knockdown experiments in D. nodosus

  • Transposon mutagenesis screening

  • CRISPR interference approaches

• Metabolic bypass evaluation:

  • Test growth rescue with glycine supplementation

  • Assess alternative one-carbon metabolism pathways

  • Determine the impact on folate cycle intermediates

Inhibitor Development Pipeline:

• High-throughput screening strategy:

  • Develop robust, scalable assays with Z' > 0.7

  • Screen diverse compound libraries (10,000-100,000 compounds)

  • Establish clear hit criteria and confirmation cascades

• Rational design approaches:

  • Structure-based virtual screening

  • Fragment-based drug discovery

  • Substrate-based inhibitor design

• Repurposing established SHMT inhibitors:

  • Modify existing human SHMT inhibitors for bacterial selectivity

  • Evaluate antimetabolites affecting related pathways

Antimicrobial Activity Evaluation:

• In vitro assessment:

  • Minimum inhibitory concentration (MIC) determination

  • Kill curve analysis

  • Resistance development frequency

• Target engagement demonstration:

  • Metabolomic changes consistent with SHMT inhibition

  • Cellular thermal shift assays to confirm target binding

  • Genetic approaches (resistant mutant generation and characterization)

• Comparative efficacy:

  • Activity against current clinical isolates

  • Combination studies with established antimicrobials

  • Efficacy in biofilm models

Translational Potential Assessment:

• In vivo efficacy in appropriate animal models

• Pharmacokinetic and pharmacodynamic studies

• Toxicity and safety evaluation

• Resistance risk assessment

This comprehensive approach will determine whether D. nodosus SHMT represents a viable antimicrobial target and guide the development of selective inhibitors.