Recombinant Shewanella halifaxensis GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is GMP synthase and what role does it play in bacterial metabolism?

XMP + ATP + Glutamine → GMP + AMP + PPi + Glutamate

In bacterial metabolism, GMP synthase plays an essential role in nucleotide biosynthesis, which is required for DNA replication, RNA synthesis, and various signaling pathways. In Shewanella species, which are known for their diverse respiratory capabilities and adaptability to various environments, purine metabolism may be particularly important for adaptation to different environmental conditions, including cold environments where many Shewanella species thrive .

How is GMP synthase structurally organized?

GMP synthase contains two functionally distinct domains that work in a coordinated manner:

• The "glutamine amide transfer" or glutaminase domain: Responsible for glutamine hydrolysis to produce the necessary amino group. This domain contains a conserved cysteine residue (Cys104 in human GMP synthase) that is essential for catalysis .

• The synthetase domain: Responsible for ATP hydrolysis and GMP formation. This domain binds XMP and ATP and catalyzes the transfer of the amino group to XMP .

These domains communicate through conformational changes, ensuring that glutamine hydrolysis is coupled to XMP amination. Inorganic pyrophosphate can inhibit the synthetase and uncouple the two domain functions, allowing glutamine hydrolysis to occur in the absence of ATP hydrolysis or GMP formation .

The functional coordination between these domains is essential for the enzyme's activity, and disruption of this communication can dramatically affect catalytic efficiency. In many bacterial species, GMP synthase functions as a dimer, and this quaternary structure is likely conserved in Shewanella halifaxensis as well .

What are the optimal storage conditions for recombinant Shewanella GMP synthase?

Based on established protocols for similar recombinant proteins from Shewanella species, the optimal storage conditions for recombinant Shewanella halifaxensis GMP synthase are:

• Short-term storage (up to one week): 4°C in appropriate buffer

• Medium-term storage (up to 6 months): -20°C in liquid form with glycerol

• Long-term storage (up to 12 months or more): -20°C or -80°C in either liquid form with 50% glycerol or lyophilized form

To maintain enzyme activity, it is critical to avoid repeated freeze-thaw cycles, as they can significantly reduce enzyme activity. The protein should be stored in small working aliquots to minimize this issue . For lyophilized protein, ensure complete reconstitution before use.

Including stabilizing agents such as glycerol (typically 5-50%) in storage buffers significantly improves stability. The standard recommendation is to add 5-50% glycerol (final concentration) when preparing the enzyme for long-term storage, with 50% being optimal for most applications .

How should recombinant Shewanella GMP synthase be reconstituted for experimental use?

Proper reconstitution of recombinant Shewanella halifaxensis GMP synthase is crucial for maintaining its activity. The recommended protocol is:

• Briefly centrifuge the vial containing the lyophilized protein prior to opening to bring the contents to the bottom of the tube .

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

• For improved stability, add glycerol to a final concentration of 5-50% (with 50% being optimal for long-term storage) .

• Gently mix the solution to ensure complete dissolution. Avoid vigorous vortexing, which can lead to protein denaturation.

• Allow the protein to sit for 10-20 minutes at 4°C to ensure complete rehydration.

• For experimental use, dilute the reconstituted protein in an appropriate reaction buffer, typically containing:

  • 50 mM Tris-HCl, pH 8.0

  • 5 mM MgCl₂

  • 1 mM DTT

  • 0.1 mM EDTA

• Prepare working aliquots to avoid repeated freeze-thaw cycles, as repeated freezing and thawing is not recommended .

For optimal activity assessment, it's recommended to test the reconstituted enzyme with a standard activity assay measuring the conversion of XMP to GMP in the presence of glutamine and ATP.

How can the glutamine hydrolysis function of GMP synthase be specifically assayed?

One effective approach utilizes inorganic pyrophosphate to uncouple the glutaminase and synthetase activities. Inorganic pyrophosphate inhibits the synthetase domain but allows glutamine hydrolysis to continue in the absence of ATP hydrolysis or GMP formation . This creates an experimental condition where only the glutaminase function is active.

Another approach involves the use of acivicin, a glutamine analog that selectively abolishes the glutaminase activity through covalent modification of a critical cysteine residue (Cys104 in human GMP synthase) . In experimental design, acivicin can be used as a negative control to confirm that observed activity is indeed from the glutaminase domain.

When ammonia is used in place of glutamine as the amino donor, acivicin has no effect on the synthetase activity . This differential response to acivicin provides a valuable tool for distinguishing between glutaminase-dependent and ammonia-dependent activities in experimental settings.

What techniques are most effective for studying the structural dynamics of GMP synthase during catalysis?

Understanding the structural dynamics of Shewanella halifaxensis GMP synthase during catalysis requires a multi-technique approach that can capture conformational changes across different timescales. Small-angle X-ray scattering (SAXS) has proven particularly valuable for studying large-scale conformational changes in multi-domain enzymes like GMP synthase .

SAXS analysis of related enzymes has revealed movement of catalytic domains away from each other while maintaining dimer interfaces, which appears to be a key mechanism for regulating enzyme activity . For example, in the case of a diguanylate cyclase-containing globin coupled sensor from Shewanella sp. ANA-3, SAXS analysis identified movement of the cyclase domains away from each other while maintaining the globin dimer interface as a potential mechanism for regulating cyclase activity .

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides complementary information about protein dynamics and solvent accessibility at the peptide level. This technique can map binding interfaces between domains, identify dynamic regions involved in catalysis, and detect allosteric effects of substrate binding.

For real-time observation of conformational changes, single-molecule Förster resonance energy transfer (smFRET) can detect distance changes between labeled domains during catalysis. This allows researchers to observe transient conformational states not visible in ensemble measurements and provides kinetic information about domain movements.

Circular dichroism (CD) spectroscopy can also be valuable for detecting secondary structure changes upon ligand binding, which has been successfully applied to study conformational changes in Shewanella proteins .

How does oxygen availability affect GMP synthase function in relation to bacterial signaling pathways?

While direct evidence linking oxygen availability to GMP synthase function in Shewanella halifaxensis is limited, there are intriguing connections between nucleotide metabolism and oxygen-sensing pathways in Shewanella species that warrant investigation.

In Shewanella sp. ANA-3, a globin coupled sensor (SA3GCS) with diguanylate cyclase activity has been shown to respond to oxygen levels and affect biofilm formation . This sensor synthesizes cyclic di-GMP (c-di-GMP), a major regulator of biofilm formation, in an oxygen-dependent manner . Specifically, binding of O₂ to the heme resulted in activation of diguanylate cyclase activity, while other ligands like NO and CO had minimal effects .

GMP synthase produces GMP, which is a precursor to GTP, which in turn is the substrate for diguanylate cyclases that produce c-di-GMP. This creates a potential linkage between GMP synthase activity and oxygen-responsive signaling pathways in Shewanella species.

Comparison of wild type Shewanella sp. ANA-3 and a deletion strain lacking the oxygen-sensing diguanylate cyclase (ΔSA3GCS) showed differences in biofilm formation, demonstrating that diguanylate cyclase activity modulates Shewanella phenotypes in response to oxygen availability . Under anaerobic conditions, wild-type Shewanella showed a 6.7-fold increase in biofilm formation compared to aerobic conditions .

This suggests that oxygen-responsive signaling through c-di-GMP may integrate with nucleotide metabolism pathways in which GMP synthase plays a key role. Further research is needed to elucidate whether GMP synthase activity in Shewanella species is directly regulated by oxygen levels or if it simply provides metabolic precursors whose flux is affected by oxygen-dependent cellular processes.

What are the key functional residues in the glutaminase domain of Shewanella GMP synthase?

Based on comparative analysis with well-characterized GMP synthases, several key functional residues in the glutaminase domain of Shewanella halifaxensis GMP synthase can be predicted. The most critical residue is likely a cysteine equivalent to Cys104 in human GMP synthase . This cysteine has been identified as the site of acivicin modification in human GMP synthase and is essential for glutamine hydrolysis .

Mass spectrometry and Edman sequence analysis have shown that Cys104 is the site of modification by acivicin in human GMP synthase . This residue is conserved among GMP synthases and is located within a predicted glutamine amide transfer domain .

The functional significance of this cysteine residue is substantial - it is involved in the hydrolysis of glutamine to produce an amino group but is not needed for the hydrolysis of ATP or amination of xanthosine 5'-monophosphate to produce GMP . This distinction highlights the specialized role of the glutaminase domain.

In addition to the catalytic cysteine, the glutaminase domain likely contains several other conserved residues that form the glutamine binding pocket and contribute to the hydrolysis mechanism. These typically include a histidine residue that functions as a general base and acidic residues that stabilize the transition state.

Site-directed mutagenesis of these predicted key residues, followed by activity assays, would be the most direct approach to confirm their roles in Shewanella halifaxensis GMP synthase.

How does Shewanella GMP synthase contribute to biofilm formation?

While there is no direct evidence specifically linking GMP synthase to biofilm formation in Shewanella halifaxensis, there are important connections between nucleotide metabolism and biofilm formation in Shewanella species that suggest potential involvement.

Biofilm formation in Shewanella is regulated by cyclic di-GMP (c-di-GMP), which is synthesized from GTP by diguanylate cyclases . GMP synthase produces GMP, which is subsequently converted to GTP, the substrate for c-di-GMP synthesis. Therefore, GMP synthase activity could indirectly affect biofilm formation by influencing the availability of GTP for c-di-GMP production.

In Shewanella sp. ANA-3, deletion of a globin-coupled sensor with diguanylate cyclase activity (ΔSA3GCS) resulted in decreased biofilm formation under both aerobic and anaerobic conditions . This demonstrates that regulation of c-di-GMP synthesis affects biofilm formation in Shewanella.

Furthermore, biofilm formation in Shewanella sp. ANA-3 shows oxygen-dependent regulation, with a 6.7-fold increase in biofilm formation under anaerobic versus aerobic conditions in wild-type strains . This oxygen-dependent response is slightly attenuated in the ΔSA3GCS strain (5.5-fold increase), suggesting that multiple regulatory mechanisms may be involved .

Based on these observations, GMP synthase could potentially influence biofilm formation in Shewanella species through several mechanisms:

• As a supplier of GMP/GTP for c-di-GMP synthesis

• As part of metabolic adaptations to different oxygen levels

• Through interactions with regulatory networks that coordinate metabolism and biofilm formation

How can transcriptional regulation studies inform our understanding of GMP synthase function in Shewanella?

Transcriptional regulation studies provide valuable insights into the functional context of GMP synthase in Shewanella species, revealing how this enzyme is integrated into cellular metabolic networks and environmental response systems.

For Shewanella genomes, regulon reconstruction has been performed for 41 transcription factors that are orthologous to previously characterized regulators . The inferred regulatory network includes genes involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses .

Understanding the transcriptional regulation of the guaA gene (encoding GMP synthase) in Shewanella would reveal:

• How GMP synthase expression is coordinated with other purine biosynthesis genes

• How its regulation responds to environmental conditions such as temperature, oxygen availability, and nutrient limitation

• Potential co-regulation with genes involved in related processes like biofilm formation

Recent work has demonstrated that integrating gene expression data with the reconstructed regulatory network helps interpret the functional significance of regulatory interactions . This approach could be applied to understand how GMP synthase expression correlates with other cellular processes in Shewanella species under various environmental conditions.

What approaches can be used to investigate potential cold adaptations in Shewanella halifaxensis GMP synthase?

As many Shewanella species are psychrophilic or psychrotolerant, their enzymes often show structural and functional adaptations for activity at lower temperatures. Several experimental approaches can be used to investigate potential cold adaptations in Shewanella halifaxensis GMP synthase:

These approaches would help elucidate how S. halifaxensis GMP synthase has adapted to function efficiently in cold environments, contributing to our understanding of psychrophilic enzyme adaptation.

How might inhibitor studies of GMP synthase contribute to antibacterial development?

GMP synthase represents a potential target for antibacterial development due to its essential role in nucleotide biosynthesis. Inhibitor studies of Shewanella halifaxensis GMP synthase could contribute to this field in several ways:

• Mechanism-based inhibitor design: Understanding the catalytic mechanism, particularly the glutamine hydrolysis function, can guide the development of mechanism-based inhibitors. Acivicin, a glutamine analog, has been shown to irreversibly inhibit GMP synthase by covalently modifying a catalytic cysteine residue (Cys104 in human GMP synthase) .

• Species-selective inhibition: Comparative analysis of Shewanella GMP synthase with human and other bacterial homologs could identify structural differences that could be exploited for selective inhibition. This is crucial for developing antibiotics with minimal side effects.

• Dual-target inhibitors: GMP synthase inhibitors could potentially be designed to simultaneously target other enzymes in purine biosynthesis, creating synergistic effects that reduce the likelihood of resistance development.

• Biofilm prevention: Given the potential connection between GMP synthase activity and biofilm formation through nucleotide signaling pathways, inhibitors might not only affect bacterial growth but also prevent biofilm formation, which is a major challenge in treating many bacterial infections.

• Cold-active enzyme targeting: If S. halifaxensis GMP synthase shows cold adaptations, these unique features could potentially be exploited for developing inhibitors specific to psychrophilic pathogens, which are increasingly recognized in healthcare settings.

These inhibitor studies would require detailed structural information, active site mapping, and understanding of species-specific enzyme characteristics, all of which would advance both basic science and applied antimicrobial research.