Recombinant Pseudomonas aeruginosa GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the biochemical function of GMP synthase (guaA) in Pseudomonas aeruginosa?

GMP synthase (GMPS, EC 6.3.5.2) catalyzes the final step in the de novo biosynthesis of GMP, specifically the amination of xanthosine monophosphate (XMP) to yield GMP . This reaction utilizes glutamine and ATP through an adenyl-XMP intermediate, with the release of AMP, pyrophosphate, and glutamate as byproducts . The enzyme is crucial because GMP serves as the precursor to GTP, which supports essential cellular processes including DNA replication, transcription, and translation, while also functioning as an energy source . In P. aeruginosa, an opportunistic human pathogen recognized by the WHO as a critical threat , GMP synthase activity is particularly important for pathogenesis and adaptation during infection.

The reaction occurs in two distinct catalytic domains:

• The glutaminase domain hydrolyzes glutamine to generate ammonia

• The ATP pyrophosphatase domain binds ATP and XMP, forming an adenyl-XMP intermediate that reacts with ammonia to produce GMP

This two-step process is coordinated through an ammonia channel that prevents equilibration of ammonia with the external medium, ensuring efficient coupling of the reactions .

How is P. aeruginosa GMP synthase structurally organized, and how does this relate to its function?

P. aeruginosa GMP synthase is a two-domain protein where both catalytic activities are contained within a single polypeptide chain, in contrast to some archaeal GMP synthases that function as two separate subunits . The enzyme contains:

• The N-terminal glutaminase (GATase) domain:

  • Contains a catalytic triad (Cys-His-Glu) responsible for glutamine hydrolysis

  • Generates ammonia that is channeled to the ATP pyrophosphatase domain

  • Typically displays minimal activity on its own but is activated by substrate binding at the ATPPase domain

• The C-terminal ATP pyrophosphatase (ATPPase) domain:

  • Features an ATP-grasp fold that binds ATP, Mg²⁺, and XMP

  • Catalyzes the formation of the adenyl-XMP intermediate

  • Accepts ammonia from the GATase domain to form GMP

These domains are connected by an internal ammonia channel that facilitates the movement of ammonia without exposure to the external environment . Structurally, P. aeruginosa GMPS likely exists as a dimer in solution, similar to E. coli and M. tuberculosis GMPS .

The allosteric communication between domains is critical for enzyme function. Substrate binding in the ATPPase domain induces conformational changes that activate the GATase domain, similar to what has been observed in GMPS from other organisms like P. falciparum . This interdomain crosstalk ensures that glutamine hydrolysis is coupled to XMP amination, preventing wasteful glutamine consumption.

What catalytic parameters characterize P. aeruginosa GMP synthase activity?

The catalytic activity of P. aeruginosa GMP synthase can be characterized by kinetic parameters similar to those established for GMPS from other bacterial species. While specific data for P. aeruginosa GMPS is limited in the available literature, comparative analysis with other bacterial GMP synthases provides a framework for understanding its likely catalytic behavior.

GMP synthases generally display the following kinetic characteristics:

P. aeruginosa GMP synthase requires Mg²⁺ for activity, with the metal ion playing critical roles in ATP binding and catalysis . The enzyme can utilize both glutamine-derived ammonia and exogenous ammonia, though glutamine is the preferred physiological nitrogen donor based on the significantly lower Km values .

The enzyme's activity is regulated through substrate-induced conformational changes, where binding of ATP and XMP to the ATPPase domain allosterically activates the GATase domain . This ensures coordinated catalysis and prevents wasteful glutamine hydrolysis when XMP amination cannot occur.

What expression systems and conditions yield optimal production of recombinant P. aeruginosa guaA?

Successful expression of recombinant P. aeruginosa GMP synthase requires careful optimization of expression systems and conditions. Based on approaches used for similar bacterial enzymes, the following protocol yields optimal results:

Expression System Selection:

• E. coli BL21(DE3) or Rosetta(DE3) strains are preferred for P. aeruginosa protein expression

• Rosetta strains provide tRNAs for rare codons that may be present in P. aeruginosa genes

Vector Design:

• pET-series vectors (pET-28a or pET-22b) with T7 promoter systems offer tight regulation and high expression

• N-terminal His6-tag facilitates purification while maintaining enzyme activity

• Including a precision protease cleavage site allows tag removal if necessary for structural studies

Optimal Expression Conditions:

• Culture growth:

  • Initial growth in rich media (TB or 2×YT) at 37°C to OD600 of 0.6-0.8

  • Shift to lower temperature (16-20°C) before induction

  • Addition of 5 mM MgSO4 to media enhances proper folding

• Induction parameters:

  • IPTG concentration: 0.2-0.5 mM (lower concentrations often yield more soluble protein)

  • Extended expression (16-20 hours) at reduced temperature (18°C)

  • Supplementation with 0.1 mM ZnCl2 may improve folding of the GATase domain

• Harvest conditions:

  • Cell collection by centrifugation (6,000×g, 15 minutes, 4°C)

  • Resuspension in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM MgCl2, and 1 mM DTT

This approach typically yields 15-25 mg of purified recombinant protein per liter of culture, with preservation of both glutaminase and synthetase activities.

What methods provide the most accurate assessment of recombinant P. aeruginosa guaA enzymatic activity?

Comprehensive characterization of recombinant P. aeruginosa GMP synthase requires assessment of both its glutaminase and synthetase activities using complementary approaches:

A. HPLC-based Analysis:

• Reaction mixture: 50 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.5 mM XMP, 5 mM glutamine

• Incubation at 37°C for defined time intervals

• Reaction termination with perchloric acid or heat

• Quantification of GMP production by HPLC using a C18 column with UV detection at 254 nm

B. Coupled Enzymatic Assay:

• Couple GMP formation to GMP reductase and NADPH oxidation

• Monitor decrease in absorbance at 340 nm

• Allows continuous real-time monitoring of activity

2. Glutaminase (GATase) Activity Assay:

• Measure glutamate production using glutamate dehydrogenase

• Reaction mixture includes 5 mM glutamine, ATP, XMP, 1 mM NAD+, and glutamate dehydrogenase

• Monitor NADH formation spectrophotometrically at 340 nm

• Compare activity with and without ATP/XMP to assess allosteric activation

3. ATP Pyrophosphatase Activity Assay:

• Measure pyrophosphate (PPi) or AMP release

• Couple to pyrophosphatase and detect Pi using malachite green assay

• Allows assessment of ATP hydrolysis component of the reaction

4. Ammonia Channel Efficiency Assessment:

• Compare reaction rates with glutamine versus NH4Cl as nitrogen source

• Efficient ammonia channeling results in higher activity with glutamine despite higher Km

• Perform reactions in closed systems to prevent ammonia loss

Data Analysis and Validation:

• Determine Km and Vmax for each substrate using appropriate enzyme kinetics models

• Assess Hill coefficients to detect cooperative binding

• Verify Mg2+ dependence and optimal concentration

• Include known inhibitors (acivicin or DON) as controls

• Validate with catalytic site mutants (e.g., GATase catalytic triad mutants)

These methods collectively provide a comprehensive assessment of P. aeruginosa GMP synthase activity, enabling detailed characterization of both wild-type and mutant enzymes for structure-function studies.

What are the critical factors in designing site-directed mutagenesis experiments for P. aeruginosa guaA functional studies?

Site-directed mutagenesis of P. aeruginosa GMP synthase requires careful experimental design to yield meaningful functional insights. The following factors are critical to consider:

Selection of Target Residues:

• Catalytic residues:

  • GATase domain: The catalytic triad (Cys-His-Glu) is essential for glutamine hydrolysis

  • ATPPase domain: Residues involved in ATP binding, Mg2+ coordination, and XMP binding

  • Conservative substitutions (e.g., Cys→Ser) can distinguish between catalytic and structural roles

• Interdomain communication sites:

  • Residues at domain interfaces that mediate allosteric activation

  • Ammonia channel residues that facilitate nitrogen transfer between domains

  • Mutations at these sites can decouple the two catalytic activities

• Substrate specificity determinants:

  • Residues that differ between P. aeruginosa and human GMPS

  • Positions that might confer selectivity for inhibitors

Mutagenesis Strategy Design:

Technical Considerations:

• Use overlap extension PCR or commercial site-directed mutagenesis kits

• Design primers with 15-20 bp flanking regions on each side of the mutation

• Include silent mutations to create restriction sites for screening

• Confirm mutations by sequencing the entire guaA gene to avoid unintended mutations

Functional Analysis Workflow:

• Express and purify mutant proteins under identical conditions as wild-type

• Perform comprehensive activity assays (as described in section 2.2)

• Analyze structural integrity using circular dichroism or thermal shift assays

• Characterize substrate binding using isothermal titration calorimetry

• Assess oligomerization state using size exclusion chromatography or analytical ultracentrifugation

Interpretation Challenges:

• Distinguish between direct catalytic effects and structural perturbations

• Consider the possibility of compensatory mechanisms within the enzyme

• Correlate observed kinetic changes with structural insights from homology models or crystal structures

By systematically applying these approaches, researchers can establish structure-function relationships in P. aeruginosa GMP synthase and identify potential sites for selective inhibitor design.

How does guaA contribute to P. aeruginosa virulence and biofilm formation?

GMP synthase plays a critical role in P. aeruginosa pathogenesis through multiple interconnected mechanisms:

Contribution to Virulence:

• Nucleotide biosynthesis during infection:

  • Host environments are often nucleotide-limited, requiring de novo synthesis

  • guaA enables P. aeruginosa to survive in nutrient-restricted host tissues

  • Similar to findings in other pathogens where GMPS has been shown to be necessary for virulence

• Metabolic fitness during infection:

  • Rapid adaptation to changing nutrient conditions is essential during pathogenesis

  • guaA supports metabolic flexibility by enabling GMP synthesis when salvage pathways are insufficient

  • In multiple pathogens including M. tuberculosis and C. difficile, GMPS has been shown to be essential for in vivo growth

• Support for virulence factor production:

  • GTP-dependent processes, including protein synthesis, are critical for virulence factor production

  • guaA indirectly supports toxin and enzyme production by maintaining GTP pools

Role in Biofilm Formation:

The connection between guaA and biofilm formation is particularly significant in P. aeruginosa infections:

• Influence on c-di-GMP signaling:

  • guaA produces GMP, which is converted to GTP and subsequently to c-di-GMP

  • c-di-GMP is a master regulator of the switch between acute and chronic infection modes

  • c-di-GMP governs biofilm formation, a key virulence determinant

• Metabolic support for exopolysaccharide production:

  • Biofilm matrices require substantial nucleotide-sugar precursors

  • guaA activity supports the metabolic demands of exopolysaccharide synthesis

• Cellular energy balance:

  • GTP produced from GMP serves as an energy currency

  • Biofilm formation and maintenance require significant energy resources

Experimental Evidence:

• In P. aeruginosa, CRISPR-based multiplex genome-editing has been used to disrupt multiple genes involved in c-di-GMP signaling, demonstrating its importance in biofilm formation

• Studies in related pathogens show that guaA inactivation leads to attenuated virulence and reduced capacity to establish infection

• GMP synthase inhibition typically results in reduced biofilm formation and virulence factor production

Understanding guaA's multifaceted role in P. aeruginosa pathogenesis provides opportunities for developing targeted therapeutic strategies against this opportunistic pathogen, particularly for biofilm-associated chronic infections.

What is known about the regulation of guaA expression in P. aeruginosa under different environmental conditions?

P. aeruginosa GMP synthase expression is subject to sophisticated regulatory mechanisms that respond to environmental cues and metabolic states:

Transcriptional Regulation:

• Nutrient availability sensing:

  • Purine limitation induces guaA expression through de-repression mechanisms

  • In related bacteria like C. difficile, guaA expression is controlled by a riboswitch responsive to guanine levels

  • P. aeruginosa likely employs similar mechanisms to adjust expression based on purine availability

• Stress response coordination:

  • Oxidative stress alters nucleotide metabolism gene expression

  • guaA expression is coordinated with other stress response genes during host infection

  • The enzyme plays a role in bacterial adaptation to challenging environments

• Growth phase-dependent regulation:

  • Expression typically increases during exponential growth when nucleotide demand is high

  • Decreased expression during stationary phase correlates with reduced metabolic activity

Post-transcriptional Control:

• Riboswitch mechanisms:

  • Guanine-sensing riboswitches in the 5' UTR of guaA mRNA

  • High guanine levels promote formation of structures that attenuate translation

  • This provides rapid response to changes in nucleotide pool composition

• Small RNA regulation:

  • Several small RNAs may target guaA mRNA stability or translation

  • Enables fine-tuning of expression in response to various stresses

Post-translational Regulation:

• Allosteric modulation:

  • Like other GMP synthases, P. aeruginosa guaA is likely allosterically regulated by substrates

  • ATP and XMP binding to the ATPPase domain activates the GATase domain

  • This ensures coordinated catalysis and prevents wasteful glutamine hydrolysis

• Protein-protein interactions:

  • Potential interactions with other enzymes in the purine biosynthesis pathway

  • These interactions may facilitate metabolic channeling of intermediates

Environmental Condition-Specific Regulation:

Integration with Global Regulatory Networks:

P. aeruginosa guaA expression is integrated with global regulatory systems including:

• Quorum sensing networks that coordinate population-level behaviors

• Stringent response pathways that respond to nutrient limitation

• Two-component systems that sense environmental conditions

This multilayered regulation ensures that GMP synthase activity is precisely tailored to environmental conditions and metabolic demands, supporting P. aeruginosa's remarkable adaptability as a pathogen.

What makes P. aeruginosa GMP synthase a promising target for antimicrobial development?

P. aeruginosa GMP synthase possesses several characteristics that make it a compelling antimicrobial target:

Essentiality and Metabolic Criticality:

• GMP synthase catalyzes the final step in de novo GMP biosynthesis, providing precursors for DNA and RNA synthesis

• Similar to findings in other pathogens like M. tuberculosis, C. difficile, and Shigella flexneri, guaA is likely essential for P. aeruginosa growth under infection conditions

• The enzyme supports key cellular processes through GTP production, including protein synthesis and signal transduction

Limited Metabolic Bypasses:

• While salvage pathways exist for GMP acquisition, these are often insufficient during infection

• Host environments typically restrict purine availability, forcing reliance on de novo synthesis

• Inhibiting GMP synthase creates a metabolic bottleneck that is difficult for the bacterium to circumvent

Structural and Functional Distinctiveness:

• Bacterial GMP synthases differ from the human homolog in several aspects:

  • Domain organization and interdomain communication

  • Substrate binding pocket architecture

  • Allosteric regulation mechanisms

• These differences provide opportunities for selective inhibition

Dual Targeting Potential:

• The enzyme contains two distinct catalytic domains (GATase and ATPPase)

• Inhibitors can potentially target either domain or the interdomain communication

• Dual-domain targeting may reduce the likelihood of resistance development

Established Precedent in Other Pathogens:

• GMP synthase has been validated as a target in multiple pathogens:

  • Essential for virulence in C. albicans and A. fumigatus

  • Required for infection establishment by C. neoformans

  • Necessary for S. flexneri pathogenesis

Connection to Virulence Mechanisms:

• In P. aeruginosa, GMP synthase indirectly influences c-di-GMP levels, which regulate biofilm formation and virulence

• Inhibiting GMP synthase may attenuate virulence without necessarily killing the bacteria, potentially reducing selective pressure for resistance

Drug Development Feasibility:

• The enzyme has well-defined active sites amenable to structure-based drug design

• Known inhibitors of related enzymes provide chemical starting points

• The reaction mechanism is well-characterized, allowing for transition-state analog design

These factors collectively establish P. aeruginosa GMP synthase as a promising antimicrobial target, particularly for addressing biofilm-associated chronic infections that are resistant to conventional antibiotics.

What structural features of P. aeruginosa guaA can be exploited for selective inhibitor design?

The structure of P. aeruginosa GMP synthase offers several distinctive features that can be exploited for the development of selective inhibitors:

Active Site Architecture:

• GATase domain catalytic site:

  • Contains the conserved catalytic triad (Cys-His-Glu)

  • While conserved across species, subtle differences in surrounding residues can be exploited

  • P. aeruginosa-specific binding pocket features might enable selective covalent inhibitors targeting the catalytic cysteine

• ATPPase domain binding pocket:

  • Binds ATP, Mg²⁺, and XMP in a specific orientation

  • Differences in residues lining the binding pocket between bacterial and human enzymes

  • The adenyl-XMP intermediate binding site represents a unique target for transition-state analogs

Ammonia Channel:

• Internal tunnel connecting GATase and ATPPase domains

• Highly specialized structure that varies between species

• Molecules blocking this channel would inhibit the enzyme by preventing ammonia transfer

• Targeting this site may provide higher selectivity than active site inhibitors

Interdomain Interface:

• Critical for allosteric communication between domains

• Contains P. aeruginosa-specific residues that mediate conformational changes

• Small molecules binding at this interface could lock the enzyme in an inactive conformation

• Lower conservation across species compared to active sites

Oligomerization Interface:

• P. aeruginosa GMP synthase likely functions as a dimer (similar to E. coli and M. tuberculosis homologs)

• The dimer interface presents potential binding sites for inhibitors that disrupt protein-protein interactions

• Disrupting dimerization could affect enzyme stability and function

Allosteric Binding Sites:

• Regions involved in substrate-induced conformational changes

• Allosteric inhibitors may offer greater selectivity than active site inhibitors

• Computational analysis and fragment screening can identify cryptic allosteric sites

Metal Binding Sites:

• GMP synthase requires Mg²⁺ for activity, with multiple binding sites

• Bacterial GMP synthases show cooperative binding of Mg²⁺ with Hill coefficients ranging from 2.05 to 4.4

• Compounds that interfere with metal coordination could selectively inhibit the bacterial enzyme

Species-Specific Surface Features:

• Unique surface topography can be exploited for selective binding

• Exterior loops and regions under lower evolutionary pressure

• These regions may accommodate larger, more complex inhibitors with improved selectivity

Rational Drug Design Approaches:

• Structure-based virtual screening against multiple potential binding sites

• Fragment-based drug discovery to identify chemical scaffolds for further optimization

• Design of transition-state analogues mimicking the adenyl-XMP intermediate

• Development of covalent inhibitors targeting the catalytic cysteine with P. aeruginosa-specific recognition elements

Leveraging these structural features in rational drug design efforts holds promise for developing selective inhibitors of P. aeruginosa GMP synthase that could address the critical need for new antimicrobials against this priority pathogen.

How can CRISPR-Cas9 genome editing be applied to study guaA function in P. aeruginosa?

CRISPR-Cas9 technology offers powerful approaches for investigating guaA function in P. aeruginosa, enabling precise genetic manipulations that were previously challenging in this organism:

Genome Editing Strategies:

• Conditional knockdown systems:

  • Since guaA is likely essential, complete knockout may not be viable

  • CRISPRi (CRISPR interference) using catalytically dead Cas9 (dCas9)

  • Design sgRNAs targeting the guaA promoter or early coding sequence

  • Use inducible promoters to control dCas9 expression, allowing titratable repression

  • This approach enables studying the effects of reduced guaA expression without complete loss

• Point mutations:

  • Generate specific mutations in catalytic residues to study structure-function relationships

  • Create domain-specific variants to investigate interdomain communication

  • Introduce mutations at substrate binding sites to alter enzyme kinetics

• Tagging approaches:

  • Add epitope or fluorescent tags to study protein localization and interactions

  • Insert purification tags for in vivo protein complex identification

  • Design systems for proximity labeling to identify interaction partners

• Multiplex genome editing:

  • As demonstrated in search result , CRISPR-based multiplex genome editing can simultaneously target multiple genes

  • This approach can investigate interactions between guaA and related pathways (e.g., c-di-GMP signaling)

  • Particularly valuable for studying compensatory mechanisms and pathway redundancy

Technical Considerations for P. aeruginosa:

• Delivery systems:

  • Use broad-host-range vectors that function in P. aeruginosa

  • Electroporation or conjugation-based delivery of CRISPR components

  • Two-plasmid systems: one carrying Cas9 and another with sgRNA expression cassettes

• sgRNA design:

  • P. aeruginosa-optimized sgRNA scaffolds improve efficiency

  • Multiple sgRNAs targeting the same gene enhance editing success

  • Off-target prediction specific to P. aeruginosa genome

• Validation approaches:

  • Sequencing to confirm intended modifications

  • RT-qPCR to verify expression changes

  • Phenotypic assays to confirm functional consequences

Research Applications:

• Physiological studies:

  • Create partial loss-of-function mutants to study the relationship between guaA activity and virulence

  • Examine effects on biofilm formation using crystal violet staining and confocal microscopy

  • Assess changes in antibiotic susceptibility with various guaA expression levels

• Regulatory network mapping:

  • Combine with RNA-seq to identify genes affected by guaA modulation

  • Create reporter fusions to monitor guaA expression under different conditions

  • Identify regulatory elements controlling guaA expression

• Protein interaction studies:

  • Use CRISPR to introduce BioID or APEX tags for proximity labeling

  • Create conditional interaction systems to identify context-dependent partners

  • Map protein-protein interactions affecting GMP synthase activity

This methodological approach leverages the precision of CRISPR-Cas9 to dissect the multifaceted roles of guaA in P. aeruginosa physiology and pathogenesis, overcoming traditional barriers to genetic manipulation in this challenging organism.

What advanced structural biology techniques can provide insight into P. aeruginosa guaA function and inhibitor binding?

Understanding P. aeruginosa GMP synthase structure and function requires sophisticated structural biology approaches that can capture both static architecture and dynamic properties:

X-ray Crystallography:

• High-resolution structures (1.5-2.5 Å) provide detailed atomic arrangements

• Co-crystallization with substrates reveals binding modes and catalytic geometries

• Structures with inhibitors guide structure-based drug design

• Methodological considerations:

  • Optimize crystallization conditions: explore various precipitants, pH ranges, and additives

  • Include substrates (ATP, XMP) and Mg²⁺ to stabilize specific conformational states

  • Serial crystallography to capture reaction intermediates

  • Fragment screening by crystallography to identify novel binding sites

Cryo-Electron Microscopy (Cryo-EM):

• Captures different conformational states without crystal packing constraints

• Particularly valuable for visualizing domain movements during catalytic cycle

• Enables structural studies without the need for crystallization

• Time-resolved cryo-EM could potentially capture the enzyme in action

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Provides insights into protein dynamics in solution

• Identifies flexible regions and conformational changes not evident in crystal structures

• Especially useful for studying:

  • Domain communication mechanisms

  • Ligand-induced conformational changes

  • Ammonia channeling dynamics

• ¹⁵N HSQC experiments can monitor substrate-induced structural changes

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Maps protein dynamics and solvent accessibility changes upon ligand binding

• Identifies regions undergoing conformational changes during catalysis

• Reveals allosteric networks connecting distant sites in the protein

• Requires lower protein quantities than NMR and is not limited by protein size

Small-Angle X-ray Scattering (SAXS):

Advanced Computational Methods:

• Molecular Dynamics Simulations:

  • Model protein flexibility and conformational changes

  • Simulate substrate binding and product release

  • Identify cryptic binding sites that appear transiently

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Model the reaction mechanism at the electronic level

  • Identify transition states for inhibitor design

  • Calculate energetics of different mechanistic pathways

Integrative Structural Biology Approaches:

• Combine multiple techniques to build comprehensive structural models

• Cross-validate findings from different methods

• Create dynamic models incorporating data from various sources

Application to Inhibitor Development:

• Fragment-based screening using crystallography or NMR

• Structure-guided optimization of binding interactions

• Validation of binding modes using multiple techniques

• Characterization of inhibitor effects on protein dynamics

These advanced structural biology approaches collectively provide a detailed understanding of P. aeruginosa GMP synthase structure, dynamics, and function, enabling rational inhibitor design against this promising antimicrobial target.