Recombinant Phenylobacterium zucineum GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the biochemical classification of Phenylobacterium zucineum GMP synthase?

Phenylobacterium zucineum GMP synthase [glutamine-hydrolyzing] (guaA) is classified as a glutamine amidotransferase (GAT) with the EC number 6.3.5.2. It catalyzes the hydrolysis of glutamine and transfers the generated ammonia to diverse metabolites. The enzyme is also known as GMP synthetase or glutamine amidotransferase and plays a crucial role in the de novo pathway for guanosine monophosphate (GMP) synthesis from xanthosine monophosphate (XMP) .

What are the optimal storage conditions for recombinant P. zucineum GMP synthase?

For optimal stability, recombinant P. zucineum GMP synthase should be stored at -20°C for regular use, or at -80°C for extended storage periods. Repeated freezing and thawing cycles should be avoided as they can compromise enzyme activity. For working solutions, aliquots can be stored at 4°C for up to one week. The shelf life of the liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form can be stable for up to 12 months under the same conditions .

What is the source organism for P. zucineum GMP synthase and what are its characteristics?

Phenylobacterium zucineum strain HLK1(T) was isolated from the human erythroleukemia cell line K562. It is a Gram-negative rod bacterium that is motile with a polar flagellum. The organism is strictly aerobic, nonfermentative, and exhibits positive oxidase and catalase activities. Its optimal growth occurs at 37°C in environments with pH between 6.5 and 7.5. Notably, P. zucineum is a facultative intracellular organism with potential pathogenic relevance to humans and mammals. The bacterium has a DNA G+C content of 71.2±0.2 mol% .

How should recombinant P. zucineum GMP synthase be reconstituted for experimental use?

For optimal reconstitution, it is recommended to briefly centrifuge the product vial prior to opening to bring contents to the bottom. The protein should be reconstituted in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL. For long-term storage stability, add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and create aliquots for storage at -20°C/-80°C. This approach minimizes protein degradation from repeated freeze-thaw cycles while maintaining enzyme activity for experimental applications .

What are the most effective methods for measuring GMP synthase activity?

GMP synthase activity can be measured using either discontinuous or continuous assay methods:

Discontinuous assays: These involve mixing the enzyme with substrate and measuring product formation after a set period. This approach is suitable for preliminary investigations or when the appropriate time interval is well-established. For GMP synthase, this could involve measuring GMP formation from XMP after a defined reaction period.

Continuous assays: These methods monitor reaction progress in real-time by continuously measuring either product appearance or substrate disappearance. For GMP synthase, this could involve spectrophotometric monitoring of nucleotide conversion. The reaction typically requires a buffer solution to maintain optimal pH (around 8.5 for many GMP synthase experiments), and should include the substrates ATP, XMP, and glutamine along with a cofactor such as Mg²⁺ .

For stopped-flow absorbance measurements specifically, a solution of GMP synthase (typically 20 μM) with ATP (2 mM) can be rapidly mixed with various concentrations of XMP. In experiments involving glutamine, XMP (120 μM) should be pre-mixed with the enzyme and ATP at room temperature for 1-3 minutes before rapid mixing with a solution containing glutamine (2 mM) and ATP in buffer containing 25 mM EPPS (pH 8.5) and 10 mM MgCl₂ .

How can researchers generate mutant variants of GMP synthase for structure-function studies?

Mutant variants of GMP synthase can be generated using site-directed mutagenesis techniques. For example, tryptophan to phenylalanine mutations can be created using the QIAGEN Quickchange site-directed mutagenesis method. This approach involves designing two complementary "mutagenizing" primers containing the desired mutation and using them to amplify the entire plasmid containing the GMP synthase gene. Including a silent mutation that adds or removes a restriction site in the primers simplifies confirmation of correct plasmid construction.

For expression of the mutant protein, the mutated plasmid can be transformed into an appropriate E. coli expression system (such as those based on pET vectors for His-tagged proteins), followed by induction, cell lysis, and purification using standard protein purification techniques .

What is the catalytic mechanism of GMP synthase and how does it coordinate its dual catalytic functions?

GMP synthase catalyzes a complex reaction involving two distinct catalytic activities that occur in separate domains but are coordinated through molecular tunneling:

• Glutaminase activity: In the glutaminase domain, glutamine is hydrolyzed to release ammonia and glutamate.

• ATPPase activity: In the ATPPase domain, XMP is converted to GMP using the ammonia generated from glutamine hydrolysis, with ATP being consumed to AMP and inorganic pyrophosphate.

The reaction proceeds via an adenylylated XMP intermediate. pH-dependent studies and ¹⁵N-edited proton NMR spectroscopy have demonstrated that ammonia released from glutamine is not equilibrated with the external medium but is instead channeled directly to the ATPPase active site through an internal tunnel connecting the two catalytic domains. Kinetic studies have shown that glutamine-dependent GMP formation is at its maximum when the ratio of glutaminase and ATPPase domains is 1:1, supporting the ammonia channeling model .

The adenylylated XMP intermediate formed during the reaction has been isolated and characterized. Mixing GMP synthase with its nucleotide substrates (ATP and XMP) in the absence of an ammonia source leads to the formation of this stable intermediate, which can be detected by HPLC. When glutamine is subsequently added to this reaction mixture, the intermediate is converted to the final product, GMP .

How does substrate binding affect the conformational dynamics of GMP synthase?

Substrate binding induces significant conformational changes in GMP synthase that are essential for its catalytic function. The binding of ATP and XMP to the ATPPase domain triggers conformational rearrangements that optimize the active site for catalysis and facilitate the formation of the adenylylated XMP intermediate.

Studies suggest that these conformational changes also play a crucial role in coordinating the activities of the glutaminase and ATPPase domains. The binding of substrates to the ATPPase domain appears to transmit allosteric signals to the glutaminase domain, enhancing its activity and ensuring that ammonia production is synchronized with the formation of the adenylylated XMP intermediate that requires the ammonia for conversion to GMP.

These conformational dynamics are essential for the efficient channeling of ammonia between the two active sites and preventing its loss to the bulk solvent. The molecular basis for this interdomain communication involves structural elements that connect the two domains and transmit information about the substrate-binding status .

What factors affect the stability and activity of recombinant GMP synthase during experimental procedures?

Several factors can influence the stability and activity of recombinant GMP synthase:

To maintain optimal enzyme activity, it is essential to minimize exposure to extreme conditions, prepare appropriate working aliquots to avoid repeated freeze-thaw cycles, and use suitable buffer systems that stabilize the protein structure while supporting catalytic activity .

How can researchers distinguish between glutamine-dependent and ammonia-dependent activities of GMP synthase?

Researchers can differentiate between glutamine-dependent and ammonia-dependent activities of GMP synthase through carefully designed experimental approaches:

• Substrate dependency studies: By conducting parallel reactions with either glutamine or ammonia (typically as ammonium salts) as the nitrogen source, researchers can directly compare the two activities. The glutamine-dependent activity requires both the glutaminase and ATPPase domains, while the ammonia-dependent activity only requires the ATPPase domain.

• pH-dependent activity analysis: Glutamine-dependent and ammonia-dependent activities often show distinct pH optima. By measuring enzyme activity across a range of pH values with either glutamine or ammonia as the nitrogen source, researchers can identify differences in pH dependency that reflect the distinct catalytic mechanisms involved.

• Isotope labeling experiments: Using ¹⁵N-labeled glutamine and monitoring the incorporation of the labeled nitrogen into GMP using NMR spectroscopy or mass spectrometry allows researchers to trace the nitrogen transfer pathway. This approach has been used to demonstrate that the ammonia released from glutamine is channeled directly to the ATPPase active site without equilibrating with the external medium .

• Domain separation or mutation: Expressing and purifying the individual domains of GMP synthase or introducing mutations that selectively disrupt one catalytic activity while preserving the other can help distinguish between the two activities. For example, mutations in the glutaminase domain may abolish glutamine-dependent activity while preserving ammonia-dependent activity .

What are the common challenges in purifying recombinant P. zucineum GMP synthase and how can they be addressed?

Purification of recombinant P. zucineum GMP synthase presents several challenges that researchers should be prepared to address:

• Expression optimization: The expression of recombinant P. zucineum proteins in E. coli may be challenging due to differences in codon usage and protein folding machinery. This can be addressed by using codon-optimized genes, expression hosts with rare tRNA supplements, or lower induction temperatures to promote proper folding.

• Solubility issues: Recombinant GMP synthase may form inclusion bodies in E. coli. Strategies to improve solubility include expression at lower temperatures (15-25°C), co-expression with molecular chaperones, or using fusion tags that enhance solubility (such as MBP or SUMO).

• Enzyme activity preservation: GMP synthase activity may be sensitive to purification conditions. Incorporating stabilizing agents such as glycerol, reducing agents, or appropriate metal ions in the purification buffers can help maintain enzyme activity.

• Purity requirements: For detailed structural and functional studies, high purity (>85% as assessed by SDS-PAGE) is essential. Multi-step purification protocols combining affinity chromatography (using His-tag), ion exchange chromatography, and size exclusion chromatography may be necessary to achieve the desired purity level .

• Storage stability: Even after successful purification, maintaining enzyme activity during storage is critical. Adding glycerol to a final concentration of 5-50%, dividing the purified protein into small working aliquots, and storing at -20°C or -80°C can help preserve enzyme activity for extended periods .

How can recombinant P. zucineum GMP synthase be used in studies of bacterial pathogenesis and host-pathogen interactions?

Recombinant P. zucineum GMP synthase represents a valuable tool for investigating bacterial pathogenesis and host-pathogen interactions due to several key factors:

• Metabolic significance: GMP synthase plays a critical role in nucleotide biosynthesis, which is essential for bacterial replication and survival within host cells. Studies using the recombinant enzyme can help elucidate how P. zucineum adapts its metabolism during intracellular growth.

• Pathogen-specific features: Comparative studies between P. zucineum GMP synthase and its human counterpart can identify structural and functional differences that might be exploited for the development of pathogen-specific inhibitors. This is particularly relevant given that P. zucineum is a facultative intracellular organism with potential pathogenic relevance to humans .

• Intracellular survival mechanisms: Since P. zucineum was isolated from a human erythroleukemia cell line (K562) , investigating the role of GMP synthase in nucleotide acquisition within host cells could provide insights into the mechanisms that enable this bacterium to survive in this niche.

• Drug target validation: Recombinant GMP synthase can be used in inhibitor screening assays to identify compounds that selectively target bacterial nucleotide biosynthesis. Such studies could contribute to the development of novel antibacterial agents against facultative intracellular pathogens.

• Host response studies: Purified recombinant GMP synthase can be used to study host immune responses to specific bacterial proteins, potentially revealing how P. zucineum interacts with human immune cells.

What techniques can be used to study the interdomain communication and ammonia channeling in GMP synthase?

Investigating interdomain communication and ammonia channeling in GMP synthase requires sophisticated biophysical and biochemical approaches:

• X-ray crystallography and cryo-EM: Structural determination of GMP synthase in different ligand-bound states can reveal conformational changes associated with interdomain communication. Comparing structures with various substrate combinations or transition state analogs can provide snapshots of the reaction pathway.

• Molecular dynamics simulations: Computational modeling based on crystal structures can simulate the movement of ammonia through the putative channel and identify key residues involved in guiding its transport between active sites.

• Site-directed mutagenesis: Systematic mutation of residues lining the predicted ammonia channel can test their importance in maintaining enzyme activity. Comparing the effects on glutamine-dependent versus ammonia-dependent activities can specifically probe the channeling function .

• Isotope labeling and NMR spectroscopy: ¹⁵N-labeled glutamine can be used in conjunction with NMR spectroscopy to track the movement of nitrogen atoms during catalysis, providing direct evidence for ammonia channeling without equilibration with the bulk solvent .

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify residues that come into proximity during the catalytic cycle, providing insights into dynamic conformational changes that facilitate interdomain communication.

• Stopped-flow kinetics: Rapid kinetic measurements can capture transient conformational states and reaction intermediates, helping to elucidate the timing of events in the coordinated reaction mechanism .

• Domain separation and complementation: Expressing the glutaminase and ATPPase domains separately and studying their ability to function together in trans versus the intact enzyme can reveal aspects of the physical coupling between domains.

How does P. zucineum GMP synthase compare to GMP synthases from other bacterial species?

P. zucineum GMP synthase shares fundamental catalytic mechanisms with GMP synthases from other bacterial species, but exhibits species-specific characteristics that reflect its evolutionary history and ecological niche:

What insights can structural biology provide about the evolution of GMP synthase in different organisms?

Structural biology offers valuable insights into the evolutionary history and functional adaptations of GMP synthase across different organisms:

• Conserved catalytic cores: Structural comparisons can reveal highly conserved catalytic cores within both the glutaminase and ATPPase domains, reflecting fundamental constraints on the chemistry of the reaction that have been maintained throughout evolution.

• Divergent peripheral regions: Areas distant from the active sites may show greater structural diversity, potentially associated with species-specific regulatory mechanisms, protein-protein interactions, or adaptations to different cellular environments.

• Domain organization: The spatial arrangement of the glutaminase and ATPPase domains and the architecture of the ammonia channel connecting them may show variations across evolutionary lineages, reflecting different solutions to the challenge of coordinating the two catalytic activities.

• Active site plasticity: Subtle differences in active site geometries may reveal adaptations to different substrate concentrations or cellular conditions encountered by various organisms in their respective ecological niches.

• Structural basis for regulation: Comparing structures of GMP synthases from diverse organisms can identify regulatory features that have evolved independently in different lineages, providing insights into the diverse strategies for controlling this important metabolic enzyme.

• Evolutionary relationship to other amidotransferases: Structural comparisons with other glutamine amidotransferases can illuminate the evolutionary history of this enzyme superfamily and the specialization of GMP synthase for its specific role in nucleotide biosynthesis .