Recombinant Yersinia pestis bv. Antiqua Glycine dehydrogenase [decarboxylating] (gcvP), partial

What is Glycine dehydrogenase [decarboxylating] (gcvP) and its role in Yersinia pestis metabolism?

Glycine dehydrogenase [decarboxylating], encoded by the gcvP gene, functions as a critical component of the glycine cleavage system (GCS) in Y. pestis. The enzyme catalyzes the degradation of glycine by binding the alpha-amino group through its pyridoxal phosphate (PLP) cofactor, resulting in the release of CO₂. The remaining methylamine moiety is subsequently transferred to the lipoamide cofactor of the H protein (GcvH) within the GCS complex . This reaction represents the first step in glycine catabolism, which plays crucial roles in one-carbon metabolism, amino acid homeostasis, and potentially pathogen survival within host environments.

The complete GCS machinery in Y. pestis consists of four components working in concert:

• GcvP (P-protein): The glycine dehydrogenase that performs the initial decarboxylation

• GcvH (H-protein): A lipoate-bearing carrier protein that accepts the methylamine moiety

• GcvT (T-protein): A tetrahydrofolate-dependent aminomethyltransferase

• GcvL (L-protein): A lipoamide dehydrogenase that regenerates the oxidized lipoic acid

This system serves to convert glycine into ammonia, carbon dioxide, and a one-carbon unit attached to tetrahydrofolate, which can then be utilized in various biosynthetic pathways.

What is the structural organization and key domains of Y. pestis gcvP?

The gcvP protein from Y. pestis bv. Antiqua (strain Angola) consists of 959 amino acids with a molecular mass of approximately 104.7 kDa . The protein exhibits a modular domain organization typical of the GcvP family, with several functionally distinct regions:

Structural studies of homologous bacterial GcvP enzymes reveal a homodimeric quaternary structure stabilized by disulfide bonds, which is likely conserved in the Y. pestis enzyme. The active site contains conserved residues for glycine binding and PLP coordination, with redox-sensitive cysteine residues implicated in regulatory disulfide formation that may respond to environmental conditions within the host.

What experimental approaches can determine gcvP activity in vitro?

To assess gcvP enzymatic activity in vitro, researchers should employ a multicomponent assay system that measures the complete glycine cleavage reaction. A robust methodological approach includes:

• Spectrophotometric coupled assay: This method monitors NAD⁺ reduction to NADH when the complete glycine cleavage system is reconstituted with purified components (gcvP, gcvH, gcvT, and dihydrolipoamide dehydrogenase). The increase in absorbance at 340 nm corresponds to the rate of glycine oxidation.

• Radiometric assay: Using ¹⁴C-labeled glycine as substrate, researchers can quantify the production of ¹⁴CO₂ by capturing evolved gas in alkaline traps followed by scintillation counting. This approach provides direct evidence of the decarboxylation activity.

• Bicarbonate incorporation assay: In the reverse reaction, gcvP can catalyze the carboxylation of methylamine-loaded gcvH. This can be measured using ¹³C-labeled bicarbonate and mass spectrometry to track isotope incorporation.

• PLP cofactor binding assay: The intrinsic fluorescence change upon PLP binding can be monitored to assess cofactor association with recombinant gcvP, providing information about the structural integrity of the catalytic site.

For optimal activity measurement, the following conditions are recommended:

• Buffer: 50 mM potassium phosphate, pH 7.2-7.5

• Temperature: 30-37°C

• Cofactors: PLP (50-100 μM), NAD⁺ (2 mM)

• Substrates: Glycine (1-10 mM), tetrahydrofolate (0.5-1 mM)

• Additional components: Lipoic acid-modified gcvH, gcvT, and dihydrolipoamide dehydrogenase

What expression systems and purification strategies yield optimal recombinant Y. pestis gcvP?

For efficient production of functional recombinant Y. pestis gcvP, researchers should consider the following optimized expression and purification protocol:

Expression System Considerations:

• Bacterial expression: E. coli BL21(DE3) or Rosetta strains carrying pET-based vectors with a C-terminal His₆-tag generally yield satisfactory quantities of soluble protein. Codon optimization for E. coli expression is recommended given the AT-rich genomic content of Y. pestis.

• Induction conditions: IPTG induction at 0.1-0.5 mM when cultures reach OD₆₀₀ of 0.6-0.8, followed by continued growth at 16-20°C for 16-18 hours significantly improves soluble protein yield compared to standard 37°C induction protocols.

• Media supplementation: Adding 50-100 μM PLP to the culture medium during induction ensures proper cofactor incorporation during protein folding.

Purification Strategy:

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin

• Intermediate purification via ion-exchange chromatography (typically Q-Sepharose)

• Polishing step using size-exclusion chromatography (Superdex 200)

The purification buffer should contain:

• 50 mM Tris-HCl or HEPES, pH 8.0

• 150-300 mM NaCl

• 5% glycerol as stabilizer

• 1 mM DTT to maintain reduced state of cysteine residues

• 20 μM PLP to ensure cofactor saturation

This approach typically yields 5-10 mg of highly purified protein per liter of bacterial culture, with >90% purity as assessed by SDS-PAGE . The recombinant protein should be immediately assessed for proper folding using circular dichroism and fluorescence spectroscopy to verify structural integrity before functional assays.

How can researchers investigate gcvP's role in Y. pestis pathogenicity?

Investigating the contribution of gcvP to Y. pestis virulence requires a multifaceted approach combining genetic, biochemical, and infection models:

Genetic Approaches:

• Gene deletion and complementation: Construction of a ΔgcvP knockout strain followed by phenotypic characterization and complementation with wild-type or mutant variants allows assessment of gcvP's role in bacterial fitness under different conditions.

• Conditional expression systems: Using tetracycline-inducible or similar regulatable promoters to control gcvP expression during different stages of infection provides temporal insights into its importance.

• Site-directed mutagenesis: Introducing mutations in key catalytic residues identified through sequence alignment with homologous enzymes can generate catalytically inactive variants while maintaining protein structure.

Biochemical and Cellular Analyses:

• Metabolomic profiling: Comparing metabolite profiles between wild-type and gcvP-deficient strains during host cell infection using LC-MS/MS can reveal alterations in glycine metabolism and connected pathways.

• Isotope tracing: Using ¹³C-labeled glycine to track carbon flux through central metabolism in the presence/absence of functional gcvP.

• Bacterial survival assays: Testing bacterial persistence within macrophages or neutrophils to assess if gcvP contributes to survival within phagocytic cells.

In vivo Models:

• Mouse infection models: Comparing virulence of wild-type and ΔgcvP strains in murine bubonic or pneumonic plague models, with assessment of bacterial burden, survival rates, and histopathological changes.

• Competitive index assays: Co-infection with wild-type and mutant strains to directly measure relative fitness in vivo.

• Transcriptional profiling: RNA-seq analysis of bacteria recovered from infected tissues to identify compensatory pathways activated in the absence of gcvP.

The combined results from these approaches can establish whether gcvP is essential for full virulence, contributes to specific aspects of pathogenesis, or represents a potential therapeutic target .

What challenges exist in structural studies of Y. pestis gcvP and how can they be addressed?

Structural characterization of Y. pestis gcvP presents several significant challenges, mainly stemming from its large size (104.7 kDa), multi-domain architecture, and requirement for cofactor binding. Researchers can address these challenges using the following approaches:

Challenges in Crystallization:

• Protein heterogeneity: The full-length gcvP protein may exhibit conformational flexibility and domain movements that impede crystal formation.

• Large size: At 959 amino acids, the complete protein may be difficult to crystallize in an ordered lattice.

• Cofactor binding: The PLP cofactor must be properly incorporated for physiologically relevant structures.

Recommended Strategies:

• Domain-based approach: Express and purify individual domains (PLP-binding domain, carrier protein interaction domain) for separate structural studies. This reduces size complexity and may improve crystallization success.

• Surface entropy reduction: Identify surface patches with high conformational entropy using computational tools and introduce mutations (e.g., Lys/Glu to Ala) to enhance crystal contacts.

• Co-crystallization strategies:

  • With PLP cofactor and substrate analogs to stabilize the active site

  • With binding partners like gcvH to capture physiologically relevant complexes

  • Using nanobodies or crystallization chaperones to minimize flexible regions

• Alternative structural methods:

  • Cryo-electron microscopy for full-length protein or complexes

  • Small-angle X-ray scattering (SAXS) to determine solution structure and domain arrangements

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics and interaction surfaces

• Crystallization condition optimization:

  • Screening with various detergents and amphiphiles as additives

  • Testing reductive environments (5-10 mM DTT) to prevent unwanted disulfide formation

  • Employing controlled proteolysis during crystallization to remove disordered regions

These approaches have proven successful for structurally characterizing related enzymes from bacterial systems and would likely yield valuable insights into the Y. pestis gcvP structure-function relationship.

How does Y. pestis gcvP compare with homologous proteins in other bacterial species?

The gcvP protein from Y. pestis exhibits significant structural and functional conservation with homologous proteins across bacterial species, while displaying specific adaptations that may relate to the pathogen's lifestyle. Comparative analysis reveals the following:

Notably, the Y. pestis enzyme contains cysteine residues at positions that enable redox regulation, similar to those documented in Rhodopseudomonas palustris. This feature may allow metabolic adaptation during transitions between environmental reservoirs and mammalian hosts, potentially contributing to pathogenicity. The enzyme also shows adaptations consistent with Y. pestis's nutritional requirements during infection cycles, including optimized activity at temperatures corresponding to mammalian host environments (37°C) compared to homologs from environmental bacteria.

What insights can ancient Y. pestis genomes provide about gcvP evolution?

Analysis of ancient Y. pestis genomes, particularly those from Late Bronze Age samples (~3800 years BP), provides valuable insights into the evolutionary trajectory of the gcvP gene in this pathogen:

Temporal Changes in gcvP Sequence:
Archaeological samples from the Samara region of Russia (~3800 BP) contain Y. pestis genomes that show distinct lineages from both Late Neolithic/Bronze Age Eurasia (LNBA, 5000-3500 BP) samples and modern strains . Comparative genomic analysis reveals several key observations about gcvP evolution:

• Sequence conservation: The core catalytic domain of gcvP shows remarkable conservation over approximately 3800 years, indicating strong selective pressure to maintain glycine decarboxylation function.

• Regulatory adaptations: Progressive changes in regulatory regions of the gcvP gene correlate with the acquisition of flea-borne transmission capability. These modifications likely altered expression patterns or regulatory responses to environmental cues encountered during the flea-mammal transmission cycle.

• Co-evolution with metabolic networks: Changes in gcvP appear coordinated with modifications in connected metabolic pathways, suggesting selection for optimized carbon and nitrogen metabolism during host adaptation.

Functional Implications:
The analysis of ancient Y. pestis gcvP sequences suggests that the refinement of glycine metabolism was an important component of the bacterium's adaptation to its current ecological niche. The gcvP enzyme likely played roles in:

• Adaptation to nutrient availability within arthropod vectors and mammalian hosts

• Nitrogen metabolism regulation during host colonization

• One-carbon unit provision for nucleotide synthesis during rapid replication phases

This evolutionary perspective provides crucial context for understanding the current function of gcvP in modern Y. pestis strains and may inform research into metabolic vulnerabilities that could be exploited for therapeutic development .

How might gcvP function contribute to Y. pestis historical evolution of virulence?

The evolution of gcvP function appears to be intertwined with Y. pestis's development as a highly virulent pathogen. Examining the role of gcvP in the context of the pathogen's evolutionary history reveals several significant contributions to virulence:

Metabolic Adaptation During Host Transition:
Y. pestis evolved from Y. pseudotuberculosis approximately 1,500-6,400 years ago, with the acquisition of new genetic elements and the loss or modification of others . During this transition from an enteric pathogen to a vector-borne pathogen, metabolic adaptations were crucial. The gcvP enzyme likely facilitated:

• Nutritional flexibility: Enhanced ability to utilize glycine as a carbon and nitrogen source in nutrient-limited host environments.

• Adaptation to hypoxic conditions: Optimization of one-carbon metabolism for survival in oxygen-limited tissues encountered during infection.

• Energy conservation: More efficient utilization of amino acids during periods of nutritional stress within the flea gut or mammalian lymphatic tissues.

Integration with Virulence Programs:
The analysis of ancient Y. pestis genomes from the Bronze Age period (~3800 BP) reveals that gcvP evolution coincided with the acquisition of key virulence traits, suggesting coordination between metabolic and virulence adaptations:

• Co-regulation with virulence factors: Transcriptional analysis suggests that gcvP expression may be coordinated with virulence programs, particularly during temperature shifts that signal host transition.

• Contribution to biofilm formation: The glycine cleavage system potentially supplies one-carbon units necessary for biofilm formation in the flea vector, a critical step in transmission.

• Support for rapid replication: Enhanced glycine metabolism through gcvP provides essential metabolic support for the rapid bacterial replication observed in bubonic and pneumonic plague.

Phylogenetic analysis indicates that several Y. pestis lineages established during the Bronze Age persist to the present day . The conservation of gcvP across these lineages suggests its fundamental importance to the pathogen's fitness, potentially making it an interesting target for therapeutic development against contemporary plague outbreaks.

What are the recommended methods for studying gcvP interactions with other glycine cleavage system components?

Investigating the interactions between gcvP and other glycine cleavage system components requires specialized methodologies that capture both structural associations and functional cooperation. The following approaches are recommended for comprehensive characterization:

Protein-Protein Interaction Analysis:

• Isothermal titration calorimetry (ITC): Provides thermodynamic parameters (ΔH, ΔS, Kd) of binding between purified gcvP and gcvH proteins. Typical experimental conditions should include:

  • Protein concentration: 10-50 μM gcvP in cell, 100-500 μM gcvH in syringe

  • Buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP

  • Temperature: 25°C with 2-3 μL injections

• Surface plasmon resonance (SPR): Offers real-time binding kinetics (kon, koff) between immobilized gcvP and flowing gcvH. Recommended setup:

  • Immobilize His-tagged gcvP on Ni-NTA sensor chip

  • Flow gcvH at 5-500 nM concentrations

  • Multi-cycle kinetics with regeneration using EDTA

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps interaction interfaces by identifying regions protected from deuterium exchange upon complex formation.

Complex Visualization:

• Cryo-electron microscopy: For structural characterization of the complete glycine cleavage complex

• Chemical cross-linking coupled with mass spectrometry: Identifies spatial proximity between specific residues in the complex

Functional Complex Reconstitution:
For functional studies, researchers should reconstitute the complete glycine cleavage system using purified components:

The reconstituted system can be monitored using:

• Spectrophotometric detection of NADH formation at 340 nm

• Coupling to 5,10-methylene-THF-dependent reactions

• Isotope-labeled glycine to track product formation by mass spectrometry

These combined approaches provide complementary information about both structural organization and functional cooperation within the glycine cleavage machinery, offering insights into potential species-specific adaptations in the Y. pestis system .

What considerations are important when designing active site mutations to study gcvP catalytic mechanism?

Designing meaningful active site mutations for mechanistic studies of Y. pestis gcvP requires careful consideration of structural conservation, catalytic requirements, and experimental readouts. The following systematic approach is recommended:

Identification of Target Residues:

• Sequence alignment analysis: Compare Y. pestis gcvP with well-characterized homologs to identify strictly conserved residues. Focus on residues within the PLP-binding domain and those interacting with glycine substrate.

• Structural homology modeling: Generate a structural model of Y. pestis gcvP based on available crystal structures of homologous enzymes to visualize the spatial arrangement of catalytic residues.

• Key residues to target:

  • The lysine residue that forms the Schiff base with PLP (typically K235 based on homology)

  • Residues coordinating the phosphate group of PLP

  • Residues forming the glycine binding pocket

  • Residues involved in proton transfer during catalysis

Mutation Design Strategy:

• Conservative substitutions: Replace catalytic residues with sterically similar but functionally distinct amino acids to parse specific contributions:

  • Lys → Arg (maintains positive charge but cannot form Schiff base)

  • Asp/Glu → Asn/Gln (eliminates negative charge while preserving hydrogen bonding)

  • His → Asn (removes imidazole ring while maintaining hydrogen bonding potential)

• Alanine scanning: Systematically replace active site residues with alanine to eliminate side chain contributions while minimizing structural perturbation.

• Non-canonical amino acid incorporation: For advanced studies, incorporate specialized amino acids (e.g., photo-crosslinkable residues) at key positions to capture transient interactions.

Experimental Validation of Mutations:

This comprehensive approach enables precise dissection of the gcvP catalytic mechanism and can reveal species-specific adaptations in the Y. pestis enzyme compared to homologs from other organisms .

What techniques are recommended for monitoring gcvP expression during Y. pestis infection cycles?

Tracking gcvP expression during Y. pestis infection cycles presents unique challenges due to the complex host environments and temporal dynamics of infection. The following methodological approaches provide complementary data on gcvP expression patterns:

In vitro Infection Model Approaches:

• Quantitative RT-PCR: Provides sensitive measurement of gcvP transcript levels from bacteria recovered from infected cells or tissues. Design primers specific to the Y. pestis gcvP gene with normalization to constitutively expressed genes like 16S rRNA or rpoD.

• RNA-seq: Offers genome-wide transcriptional profiling to place gcvP expression in the context of global metabolic adaptations. Specialized protocols for bacterial RNA extraction from host tissues include:

  • Differential lysis approaches to selectively release bacterial RNA

  • Ribo-depletion specific for both bacterial and host rRNA

  • Unique molecular identifiers (UMIs) to control for amplification bias

• Translational reporter fusions: gcvP promoter-GFP or -luciferase constructs integrated into the Y. pestis genome enable real-time monitoring of gene expression. Flow cytometry can then quantify expression at the single-cell level from recovered bacteria.

Advanced In vivo Monitoring Systems:

• In vivo expression technology (IVET): Identify conditions where gcvP is specifically induced during infection using antibiotic resistance or nutritional selection markers.

• Recombinase-based in vivo expression technology (RIVET): Provides temporal resolution of gene induction through irreversible genetic changes.

• Dual-fluorescent protein timer constructs: Express fast-maturing and slow-maturing fluorescent proteins from the gcvP promoter to distinguish between current and historical expression levels.

Protein-Level Detection Methods:

• Targeted proteomics: Using multiple reaction monitoring (MRM) mass spectrometry with isotopically labeled peptide standards derived from gcvP to quantify protein levels directly from infection samples.

• Proximity labeling approaches: Express gcvP fused to biotin ligase (TurboID) to biotinylate proximal proteins under specific infection conditions, providing insights into protein-protein interactions during infection.

Experimental Design Considerations:
For comprehensive profiling of gcvP expression, samples should be collected at key infection timepoints:

• Early infection (first 6-12 hours): Adaptation to host environment

• Mid infection (24-48 hours): Bacterial replication phase

• Late infection (72+ hours): Persistence and transmission preparation

Additionally, comparisons between different infection models (bubonic vs. pneumonic) and different host tissues (lymph node, spleen, lung) can reveal condition-specific regulation of gcvP expression relevant to pathogenesis .

How might understanding gcvP function contribute to novel antimicrobial strategies?

The glycine dehydrogenase (gcvP) presents several promising avenues for antimicrobial development against Y. pestis, based on its essential metabolic functions and structural features:

Targetable Vulnerabilities:

• PLP-binding pocket: The pyridoxal phosphate binding site of gcvP contains specific residues and structural features that differ from mammalian glycine decarboxylating enzymes. These differences can be exploited to design selective inhibitors that disrupt cofactor binding.

• Protein-protein interaction interfaces: The interaction between gcvP and gcvH represents a potentially druggable interface. Small molecules or peptide mimetics that disrupt this interaction could specifically inhibit the glycine cleavage system without directly targeting enzymatic activity.

• Allosteric regulation sites: Comparative analysis suggests the presence of Y. pestis-specific regulatory regions within gcvP that respond to metabolic status. These allosteric sites represent opportunities for species-selective targeting.

Drug Development Strategies:

• Mechanism-based inhibitors: Design of transition-state analogs that mimic the decarboxylation intermediate could yield potent inhibitors. Computational docking studies suggest several scaffolds with potential for development:

  • Phosphono-glycine derivatives

  • Glycine analogs with electron-withdrawing groups

  • Heterocyclic compounds that mimic the PLP-glycine adduct

• Fragment-based drug discovery: Screen fragment libraries against purified gcvP to identify binding hotspots that can be developed into lead compounds with enhanced selectivity for bacterial over mammalian enzymes.

• Metabolic bypass inhibition: Combined targeting of gcvP along with redundant metabolic pathways could enhance efficacy by preventing compensatory metabolism.

Potential Impact on Y. pestis Infection:
Inhibition of gcvP function could potentially:

• Disrupt bacterial adaptation to the host environment

• Impair survival under glycine-rich or glycine-limited conditions

• Reduce virulence factor expression through metabolic stress

• Enhance susceptibility to existing antibiotics through metabolic dysregulation

Current research gaps include validation of gcvP essentiality in various infection models and demonstration of the metabolic consequences of gcvP inhibition in vivo. Future studies should focus on genetic validation (using conditional knockdowns) and chemical validation (using available tool compounds) to establish proof-of-concept for gcvP-targeted antimicrobial strategies .

What are the current technical limitations in studying Y. pestis gcvP and how might they be overcome?

Current research on Y. pestis gcvP faces several technical challenges that limit comprehensive characterization. Understanding these limitations and implementing strategies to overcome them will advance our knowledge of this important metabolic enzyme:

Current Technical Limitations:

• Biosafety restrictions: As Y. pestis is a Tier 1 Select Agent, working with native protein requires specialized containment facilities, limiting widespread research.

• Complex multiprotein system: The glycine cleavage system requires four coordinated components (gcvP, gcvH, gcvT, and dihydrolipoamide dehydrogenase), making reconstitution challenging.

• Post-translational modifications: The lipoylation of gcvH, essential for system function, is difficult to achieve in heterologous expression systems.

• Structural characterization barriers: The large size (104.7 kDa) and multi-domain nature of gcvP create challenges for crystallization and structural determination.

• Limited in vivo models: Studying gcvP function during authentic infection cycles requires specialized animal models and facilities for Y. pestis.

Strategic Approaches to Overcome Limitations:

• Surrogate systems development:

  • Express recombinant Y. pestis gcvP in attenuated strains or closely related non-pathogenic Yersinia species

  • Develop cell-free transcription-translation systems loaded with Y. pestis components

  • Create chimeric proteins with domains from Y. pestis gcvP in non-pathogenic bacterial scaffolds

• Advanced protein engineering:

  • Design minimal functional constructs focusing on the catalytic domain

  • Incorporate unnatural amino acids at key positions for mechanistic studies

  • Employ sortase-mediated ligation to combine separately produced domains

• Cutting-edge structural biology approaches:

  • Cryo-electron microscopy for large complexes

  • Integrative structural modeling combining low-resolution data

  • AlphaFold2 and similar AI tools for structure prediction validated by experimental data

• System reconstitution strategies:

  • Co-expression of all glycine cleavage components in E. coli

  • Enzymatic lipoylation of gcvH in vitro using purified lipoyl ligase

  • Nanodisc incorporation for membrane-associated studies

• Alternative infection models:

  • Tissue-engineered human systems (organoids)

  • Drosophila infection models for initial screening

  • Cell culture infection systems with fluorescent reporters

These approaches can collectively address the technical barriers while maintaining appropriate biosafety measures. Additionally, the development of biosafe surrogate systems would democratize research on Y. pestis gcvP metabolism, potentially accelerating discovery of novel antimicrobial targets .

How can researchers effectively use isotope labeling to track gcvP-mediated carbon flux?

Isotope labeling represents a powerful approach for investigating gcvP-mediated carbon flux in Y. pestis metabolism. These techniques can elucidate the integration of glycine metabolism with broader metabolic networks during infection and adaptation. The following methodological framework is recommended:

Isotope Selection and Experimental Design:

• ¹³C-labeled glycine: Using site-specifically labeled glycine (e.g., [1-¹³C]glycine or [2-¹³C]glycine) enables tracking of carbon atoms through distinct metabolic fates:

  • [1-¹³C]glycine: Tracks carbon released as CO₂ during gcvP-mediated decarboxylation

  • [2-¹³C]glycine: Follows the methylene group transferred to tetrahydrofolate

• ¹⁵N-labeled glycine: Allows tracking of nitrogen incorporation into downstream metabolites and distinction between intact glycine utilization versus gcvP-mediated cleavage.

• Dual isotope approaches: Combining ¹³C and ¹⁵N labeling provides comprehensive pathway mapping through detection of metabolism-specific isotopomer patterns.

Analytical Methodologies:

• Gas chromatography-mass spectrometry (GC-MS): Particularly effective for analyzing volatile metabolites and derivatized intermediates from central carbon metabolism.

  • Sample preparation: Acidic extraction followed by derivatization (often with MSTFA or MCF)

  • Analysis parameters: Temperature gradient from 60-320°C on a DB-5MS column

  • Data processing: Correction for natural isotope abundance using established algorithms

• Liquid chromatography-mass spectrometry (LC-MS): Preferred for polar and non-volatile metabolites like amino acids, nucleotides, and cofactors.

  • Separation: HILIC chromatography for polar metabolites

  • Detection: High-resolution MS with polarity switching

  • Quantification: Use of internal standards with matched isotope labeling

• Nuclear magnetic resonance (NMR) spectroscopy: Provides positional isotope enrichment information without derivatization.

  • ¹³C-NMR: Direct observation of labeled carbon positions

  • ¹H-¹³C HSQC: Enhanced sensitivity for detecting ¹³C incorporation

Flux Analysis Implementation:
For comprehensive understanding of gcvP's role in metabolic networks, isotope data should be integrated into metabolic flux analysis:

• Steady-state ¹³C metabolic flux analysis (¹³C-MFA):

  • Culture bacteria with ¹³C-labeled glycine under controlled conditions

  • Measure isotopomer distributions in metabolic intermediates

  • Apply computational modeling to estimate flux distributions

• Dynamic labeling experiments:

  • Switch from unlabeled to labeled substrate and monitor time-dependent label incorporation

  • Provides information on pathway dynamics and metabolite turnover rates

• In vivo applications:

  • Infect cell cultures or animal models with Y. pestis

  • Administer isotope-labeled glycine during infection

  • Recover bacteria from tissues and analyze isotope distribution

This integrated approach provides quantitative insights into how Y. pestis utilizes the glycine cleavage system during various growth conditions and infection stages. The resulting flux maps can identify metabolic vulnerabilities and adaptations specific to Y. pestis pathogenesis .