Recombinant Yersinia pseudotuberculosis serotype O:3 GMP synthase [glutamine-hydrolyzing] (guaA), partial

Structural and Functional Fundamentals

• What is GMP synthase and what role does it play in Y. pseudotuberculosis metabolism?

GMP synthase [glutamine-hydrolyzing] (guaA) is a critical enzyme in the de novo purine nucleotide biosynthesis pathway. It catalyzes the conversion of xanthosine monophosphate (XMP) to guanosine monophosphate (GMP) through the amination of XMP, utilizing glutamine as the nitrogen donor. This reaction requires ATP and results in the production of AMP and inorganic pyrophosphate as byproducts. The enzyme is essential for guanine nucleotide production, which subsequently impacts numerous cellular processes including DNA/RNA synthesis and signal transduction .

• What are the key structural domains of GMP synthase and how do they contribute to its function?

GMP synthase typically contains two major functional domains:

The coordinated action of these domains enables the enzyme to hydrolyze glutamine and transfer the generated ammonia to XMP, ultimately forming GMP .

• How is recombinant Y. pseudotuberculosis GMP synthase typically produced and prepared for research?

Recombinant Y. pseudotuberculosis GMP synthase is typically produced in E. coli expression systems. Based on protocols for similar recombinant proteins, the production process involves:

• Gene cloning into appropriate expression vectors

• Transformation into E. coli host cells

• Protein expression induction

• Cell lysis and protein purification (often yielding >85% purity by SDS-PAGE)

• Storage in appropriate buffer conditions

For reconstitution of lyophilized protein, centrifugation of the vial is recommended prior to opening. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of glycerol (5-50% final concentration) for long-term storage at -20°C/-80°C .

Basic Research Methodologies

• What are the optimal storage and handling conditions for recombinant GMP synthase?

Repeated freezing and thawing cycles should be avoided as they may compromise protein stability and activity. The general shelf life for liquid form is approximately 6 months at -20°C/-80°C, while lyophilized preparations can be stored for up to 12 months at the same temperatures .

• How can I verify the enzymatic activity of recombinant GMP synthase?

Several experimental approaches can be employed to assess GMP synthase activity:

• Spectrophotometric assays monitoring the conversion of XMP to GMP

• Coupled enzyme assays linking GMP production to a detectable signal

• HPLC or mass spectrometry to measure substrate consumption and product formation

• pH-dependent activity studies comparing glutamine-dependent and ammonia-dependent activities

• ¹⁵N-edited proton NMR spectroscopy to track ammonia channeling from glutamine to the final GMP product

Research data indicates that Gln-dependent GMP formation is maximal when the ratio of GATase and ATPPase domains/subunits is 1:1, supporting the ammonia channeling mechanism .

• What experimental approaches are recommended for studying GMP synthase's role in bacterial physiology?

For examining potential connections to biofilm formation, researchers should consider quantifying poly-β-1,6-N-acetylglucosamine production and measuring cyclic di-GMP levels, as these pathways may be indirectly influenced by GMP synthase activity .

Advanced Research Questions

• How does ammonia channeling occur within the GMP synthase enzyme complex?

Ammonia channeling is a sophisticated process in GMP synthase that prevents the equilibration of ammonia with the external medium. pH-dependent studies of glutamine- and ammonia-dependent activities, coupled with ¹⁵N-edited proton NMR spectroscopy, have established that ammonia released from glutamine is channeled directly to the ATPPase active site .

The channeling mechanism involves:

• Glutamine hydrolysis at the GATase domain catalytic triad

• Formation of a protected hydrophobic tunnel connecting the two active sites

• Transport of ammonia through this tunnel to the ATPPase domain

• Nucleophilic attack of ammonia on the adenyl-XMP intermediate

• GMP formation without ammonia release into the surrounding environment

This interconnected process ensures efficient catalysis and prevents the loss of reactive ammonia, representing a hallmark example of substrate channeling in multi-domain enzymes.

• What is the molecular basis for allosteric regulation in GMP synthase?

Allosteric regulation in GMP synthase coordinates the activities of its two catalytic domains. In the GMP synthases studied thus far, the GATase domain/subunit is typically inactive or weakly active on its own. The binding of ATP·Mg²⁺ and XMP to the ATPPase domain allosterically activates the GATase domain, enabling glutamine binding and hydrolysis .

This regulatory mechanism involves:

• Conformational changes transmitted between domains upon substrate binding

• Formation of the ammonia channel only when both domains are properly aligned

• Coordinated catalysis ensuring that glutamine hydrolysis occurs only when the ATPPase domain is ready to utilize the generated ammonia

This sophisticated allosteric control prevents wasteful glutamine consumption and ensures efficient coupling of the two catalytic reactions .

• How might post-translational modifications affect GMP synthase activity in pathogenic contexts?

While the search results don't specifically address post-translational modifications (PTMs) of Y. pseudotuberculosis GMP synthase, advanced research would investigate:

• Phosphorylation sites that might modulate catalytic activity or protein-protein interactions

• Acetylation patterns that could affect allosteric regulation

• Potential redox-sensitive residues that might respond to host-induced oxidative stress

• Proteolytic processing that might alter enzyme activity or localization

These modifications could serve as regulatory mechanisms adapting GMP synthase activity to changing environmental conditions during infection or biofilm formation.

Comparative and Evolutionary Aspects

• How does GMP synthase vary between Y. pseudotuberculosis and closely related species?

While specific comparative data for GMP synthase across Yersinia species is limited in the search results, evolutionary patterns can be inferred from related research on these pathogens:

Y. pestis evolved from Y. pseudotuberculosis with "a significant reduction in the complexity of its c-di-GMP signalling network" . This evolutionary shift likely reflects the different disease cycles of these human pathogens. Since GMP synthase produces a precursor for GTP, which is subsequently used for c-di-GMP synthesis, there may be corresponding adaptations in GMP synthase regulation or activity between these species.

The core catalytic function of GMP synthase is likely conserved across Yersinia species due to its essential metabolic role, but regulatory mechanisms may differ to accommodate the distinct lifestyles of these pathogens .

• What is the relationship between GMP synthase activity and bacterial adaptation to different environments?

GMP synthase activity is intrinsically linked to bacterial adaptation through its impact on guanine nucleotide availability. In Y. pseudotuberculosis, which transitions between environmental reservoirs, insect vectors, and mammalian hosts, nucleotide metabolism must adapt to diverse nutritional conditions.

The regulatory protein RovM serves as a molecular switch coordinating biofilm formation and motility in response to nutrient availability in Y. pseudotuberculosis . While not directly connected to GMP synthase in the search results, this illustrates how metabolic sensing (potentially including nucleotide synthesis pathways) influences key adaptation mechanisms.

As nucleotide synthesis is energy-intensive, tight regulation of GMP synthase activity would be expected during transitions between nutrient-rich and nutrient-limited environments, potentially affecting virulence and persistence .

Pathogenicity and Virulence Connections

• How might GMP synthase inhibition affect Y. pseudotuberculosis virulence?

Inhibition of GMP synthase would likely impact Y. pseudotuberculosis virulence through several mechanisms:

• Disruption of nucleotide pools essential for bacterial replication during infection

• Potential reduction in GTP availability, affecting protein synthesis and energy metabolism

• Possible indirect effects on cyclic di-GMP signaling, which regulates biofilm formation

Research on the related pathogen Y. pestis provides relevant insights: while a mutant incapable of c-di-GMP synthesis was unaffected in virulence, an hmsP mutant (unable to degrade c-di-GMP) showed reduced virulence in subcutaneous infection models due to poly-β-1,6-N-acetylglucosamine overproduction . This suggests that disruption of nucleotide-related signaling pathways can significantly impact virulence, making GMP synthase a potential therapeutic target.

• What is the connection between GMP synthase, cyclic di-GMP signaling, and biofilm formation?

The connection between GMP synthase, cyclic di-GMP signaling, and biofilm formation involves multiple layers:

In Y. pseudotuberculosis, the LysR-type regulator RovM inversely regulates biofilm formation and motility by acting as a transcriptional regulator . Under nutrient-limited conditions, RovM represses β-GlcNAc production by negatively regulating hmsHFRS expression through direct binding to the promoter region .

This regulatory network highlights how nucleotide metabolism (including GMP synthesis) is integrated with environmental sensing and biofilm regulation, ultimately affecting bacterial virulence and persistence.

Advanced Experimental Design

• How can researchers design experiments to investigate the role of GMP synthase in Y. pseudotuberculosis pathogenesis?

A comprehensive experimental design for investigating GMP synthase in pathogenesis would include:

• Generation of conditional guaA mutants (complete knockouts may be lethal)

• Construction of site-directed mutants targeting:

  • Catalytic triad residues in the GATase domain

  • ATP and XMP binding sites in the ATPPase domain

  • Residues lining the putative ammonia channel

• Infection models to assess:

  • Bacterial survival and replication in macrophages

  • Colonization efficiency in animal models

  • Competition assays between wild-type and mutant strains

• Molecular analyses:

  • Transcriptomic and proteomic profiling under infection-relevant conditions

  • Metabolomic analysis of nucleotide pools

  • Quantification of cyclic di-GMP levels and biofilm formation capacity

This multifaceted approach would provide insights into both the fundamental biochemistry of GMP synthase and its significance in bacterial pathogenicity .

• What techniques can be used to identify potential inhibitors of Y. pseudotuberculosis GMP synthase?

For lead compound optimization, researchers should consider:

• Domain-specific inhibitors targeting either GATase or ATPPase functions

• Compounds disrupting allosteric communication between domains

• Molecules blocking the ammonia channel

• Selective inhibitors with limited activity against human homologs

This research approach could potentially yield novel antimicrobial strategies against Y. pseudotuberculosis infections.