Recombinant Stenotrophomonas maltophilia GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is GMP synthase (guaA) and what is its primary function in Stenotrophomonas maltophilia?

GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2), encoded by the guaA gene in Stenotrophomonas maltophilia, is a critical enzyme in the de novo purine biosynthesis pathway. It catalyzes the amination of xanthosine monophosphate (XMP) to yield guanosine monophosphate (GMP), utilizing glutamine and ATP through an adenyl-XMP (AMP-XMP) intermediate . This reaction is essential for guanine nucleotide biosynthesis, which is required for DNA and RNA synthesis, signaling pathways, and energy metabolism in S. maltophilia.

The enzyme belongs to the class I triad amidotransferase family (G-type) and features two distinct catalytic domains that perform separate chemistries:

• The glutaminase or glutamine amidotransferase (GATase) domain - responsible for glutamine hydrolysis

• The ATP pyrophosphatase (ATPPase) domain - responsible for GMP formation

In bacterial systems like S. maltophilia, these two functional units exist as domains within a single polypeptide chain, contrasting with archaeal systems where they often exist as separate protein subunits encoded by different genes .

How does the partial recombinant GMP synthase differ from the native enzyme?

The partial recombinant Stenotrophomonas maltophilia GMP synthase differs from the native enzyme in several key aspects:

The partial recombinant form is produced specifically for research applications and may contain only the functional domains necessary for specific experimental investigations. While this approach facilitates protein production and purification, researchers should consider the potential limitations in enzymatic function when using partial constructs compared to the full-length protein.

What are the optimal storage and handling conditions for the recombinant S. maltophilia GMP synthase?

For optimal stability and activity of recombinant S. maltophilia GMP synthase, the following storage and handling protocols are recommended:

Storage Conditions:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C or -80°C to maintain stability

• Avoid repeated freeze-thaw cycles as this significantly reduces enzymatic activity

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (recommended: 50%) to prevent freeze damage during storage

• Prepare small aliquots to minimize freeze-thaw cycles

Shelf Life Expectations:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

What methods are recommended for assessing the enzymatic activity of recombinant GMP synthase?

For comprehensive assessment of recombinant GMP synthase activity, several complementary methodological approaches are recommended:

Spectrophotometric Assays:

• Glutamine-Dependent Activity: Monitor the formation of GMP by measuring the increase in absorbance at 290 nm, which reflects the conversion of XMP to GMP

• ATPase Activity: Quantify inorganic phosphate release using malachite green or similar phosphate detection reagents

• Glutaminase Activity: Measure glutamate production using glutamate dehydrogenase coupled assays

Advanced Analytical Methods:

• HPLC Analysis: Separate and quantify reaction substrates (XMP, ATP, glutamine) and products (GMP, AMP, PPi, glutamate) using ion-exchange or reverse-phase HPLC

• Mass Spectrometry: Detect the formation of the adenyl-XMP intermediate and final GMP product

• 15N-NMR Spectroscopy: Track ammonia transfer from glutamine to XMP, particularly useful for studying ammonia channeling

Experimental Conditions for Activity Assays:

• Buffer: 50 mM Tris-HCl (pH 7.5-8.0)

• Required components: ATP, MgCl₂, XMP, L-glutamine

• Typical concentrations: 2-5 mM ATP, 5-10 mM MgCl₂, 0.5-1 mM XMP, 5-10 mM L-glutamine

• Allosteric activators: ATP·Mg²⁺ and XMP (pre-incubation may enhance GATase activity)

Researchers should include appropriate controls to distinguish between glutamine-dependent and ammonia-dependent GMP formation, as the enzyme can utilize external ammonia in the absence of glutamine hydrolysis .

What are the key structural domains of GMP synthase and how do they contribute to the enzyme's function?

GMP synthase from S. maltophilia, like other bacterial GMP synthases, contains two major structural domains with distinct functions that work cooperatively through sophisticated molecular mechanisms:

1. Glutaminase (GATase) Domain:

• Contains the catalytic triad (Cys-His-Glu) typical of class I amidotransferases

• Responsible for glutamine hydrolysis generating ammonia

• Generally inactive or weakly active in isolation

• Requires allosteric activation by the ATPPase domain upon substrate binding

2. ATP Pyrophosphatase (ATPPase) Domain:

• Binds ATP·Mg²⁺ and XMP

• Catalyzes the formation of adenyl-XMP intermediate

• Contains the ammonia binding site where the nucleophilic attack on adenyl-XMP occurs

• Functions as both a synthetase and an allosteric activator of the GATase domain

Interdomain Connection:

• A molecular tunnel connects the two active sites, facilitating ammonia channeling

• The tunnel prevents the equilibration of ammonia with the external medium

• This channeling ensures efficient coupling between glutamine hydrolysis and GMP formation

Allosteric Regulation Mechanism:
The binding of ATP·Mg²⁺ and XMP to the ATPPase domain induces conformational changes that propagate to the GATase domain, activating glutamine binding and hydrolysis. This sophisticated allosteric mechanism ensures that glutamine is hydrolyzed only when the acceptor substrate (XMP) is available, preventing wasteful glutamine consumption .

How does the ammonia channeling mechanism work in GMP synthase, and what techniques can be used to study it?

The ammonia channeling mechanism in GMP synthase represents a sophisticated example of substrate tunneling that connects two spatially distinct active sites:

Ammonia Channeling Mechanism:

• Glutamine binds to the GATase domain and undergoes hydrolysis to generate ammonia

• The ammonia molecule travels through an intramolecular tunnel approximately 20-25 Å long

• The tunnel shields ammonia from the bulk solvent, preventing protonation to ammonium

• The uncharged ammonia reaches the ATPPase active site where it acts as a nucleophile

• Ammonia attacks the adenyl-XMP intermediate, displacing AMP and forming GMP

Evidence for Ammonia Channeling:

• pH-dependent studies show that glutamine-dependent activity is optimal at neutral pH, while ammonia-dependent activity requires higher pH (where NH₃ predominates over NH₄⁺)

• ¹⁵N-edited proton NMR spectroscopy experiments with labeled glutamine demonstrate that ammonia from glutamine is not equilibrated with the external medium

• Kinetic studies with two-subunit GMPS show maximal activity at a 1:1 ratio of GATase to ATPPase, supporting direct transfer of ammonia

Methodologies to Study Ammonia Channeling:

• Site-Directed Mutagenesis: Identify residues lining the putative tunnel and evaluate the effect of mutations on coupled activity

• Isotope Labeling: Use ¹⁵N-labeled glutamine and track the incorporation of labeled nitrogen into GMP

• Kinetic Isotope Effects: Compare reaction rates with ¹⁵N vs. ¹⁴N glutamine

• Cryogenic Electron Microscopy: Capture conformational changes associated with channel formation

• Molecular Dynamics Simulations: Model ammonia movement through the predicted tunnel

Understanding the ammonia channeling mechanism provides insights into the enzyme's efficiency and offers potential targets for inhibitor design in antimicrobial research.

How can recombinant GMP synthase be used to study allosteric regulation in multi-domain enzymes?

Recombinant GMP synthase from S. maltophilia provides an excellent model system for investigating allosteric regulation in multi-domain enzymes due to its well-defined domain structure and inter-domain communication:

Experimental Approaches to Study Allosteric Regulation:

• Domain Separation Studies:

  • Express and purify individual GATase and ATPPase domains separately

  • Compare activities of isolated domains versus the intact enzyme

  • Reconstitute activity by mixing domains in different ratios

  • This approach allows identification of which structural elements are necessary for allosteric communication

• Substrate-Induced Conformational Changes:

  • Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map regions affected by substrate binding

  • Apply fluorescence resonance energy transfer (FRET) by labeling specific residues in each domain to measure conformational changes in real-time

  • Crystallize the enzyme in different substrate-bound states to capture structural transitions

• Signal Propagation Pathway Identification:

  • Perform alanine-scanning mutagenesis along the inter-domain interface

  • Use double-mutant cycle analysis to identify energetically coupled residues

  • Apply computational approaches such as statistical coupling analysis (SCA) to identify co-evolving networks of amino acids

• Real-Time Monitoring of Allosteric Activation:

  • Develop biosensors using environmentally sensitive fluorophores at key positions

  • Monitor changes in intrinsic tryptophan fluorescence upon substrate binding

  • Apply single-molecule FRET to observe individual molecules transitioning between states

This research has broader implications for understanding allosteric regulation in other multi-domain enzymes and developing strategies to modulate enzyme function through allosteric sites rather than active sites.

What are the approaches for engineering GMP synthase variants with modified substrate specificity or enhanced catalytic efficiency?

Engineering GMP synthase variants offers potential for creating enzymes with novel properties for both fundamental research and biotechnological applications. Several strategies can be employed:

Rational Design Approaches:

• Active Site Engineering:

  • Identify residues directly interacting with substrates through structural analysis

  • Introduce mutations that alter the size, shape, or electrostatic properties of the binding pocket

  • Target residues that coordinate metal ions or participate in transition state stabilization

• Substrate Tunnel Modification:

  • Alter the dimensions or hydrophobicity of the ammonia channel to accommodate different substrates

  • Introduce mutations that modify the dynamics of channel opening/closing

  • Engineer gating residues to control substrate access

• Domain Interface Engineering:

  • Modify the communication between domains to enhance allosteric activation

  • Alter the flexibility of interdomain linkers to optimize domain positioning

  • Introduce new interdomain interactions to stabilize catalytically active conformations

Directed Evolution Strategies:

• Library Construction Methods:

  • Error-prone PCR targeting the entire gene or specific domains

  • DNA shuffling between GMP synthases from different species

  • Site-saturation mutagenesis at hotspot residues identified from structural analysis

• Selection/Screening Systems:

  • Develop growth-based selection in auxotrophic strains lacking endogenous GMP synthase

  • Create high-throughput colorimetric or fluorescent assays for detecting product formation

  • Implement phage display or yeast surface display coupled with activity-based probes

Example Modification Targets for Enhanced Properties:

These approaches can lead to GMP synthase variants with novel properties for applications in nucleotide analog synthesis, biosensors, or therapeutic enzyme development.

How can recombinant GMP synthase be used to develop potential therapeutic strategies against S. maltophilia infections?

Recombinant GMP synthase offers several promising avenues for developing therapeutic strategies against difficult-to-treat S. maltophilia infections:

Target Validation and Inhibitor Discovery:

• High-Throughput Screening Platforms:

  • Use purified recombinant GMP synthase to screen chemical libraries for inhibitors

  • Develop fluorescence-based or colorimetric assays suitable for 96/384-well plate formats

  • Prioritize compounds that selectively inhibit bacterial GMP synthase over human homologs

• Structure-Based Drug Design:

  • Utilize crystal structures of S. maltophilia GMP synthase to design targeted inhibitors

  • Focus on unique features of the bacterial enzyme, particularly the ammonia channel and allosteric sites

  • Apply computational methods including molecular docking and virtual screening to identify lead compounds

• Fragment-Based Approaches:

  • Screen fragment libraries against different domains of GMP synthase

  • Link or grow fragments that bind to adjacent pockets

  • Develop bivalent inhibitors that target both the GATase and ATPPase domains

Therapeutic Applications Beyond Direct Inhibition:

• Anti-Biofilm Strategies:

  • Develop compounds that modulate GMP synthase activity to disrupt biofilm formation

  • Target the connection between guanine nucleotide metabolism and c-di-GMP signaling

  • Create combination therapies that inhibit GMP synthase while disrupting established biofilms

• Vaccine Development:

  • Evaluate recombinant GMP synthase as a potential vaccine antigen

  • Identify immunogenic epitopes that can stimulate protective immunity

  • Develop attenuated S. maltophilia strains with modified GMP synthase activity

• Diagnostic Applications:

  • Develop antibodies against GMP synthase for diagnostic tests

  • Create biosensors using recombinant GMP synthase to detect specific inhibitors or antibodies

  • Establish point-of-care tests for rapid identification of S. maltophilia infections

These approaches are particularly valuable considering that S. maltophilia is an opportunistic pathogen intrinsically resistant to multiple and broad-spectrum antibiotics, making it difficult to eliminate using conventional treatment methods .

What are common challenges in expressing and purifying recombinant S. maltophilia GMP synthase, and how can they be addressed?

Researchers often encounter several challenges when working with recombinant S. maltophilia GMP synthase. Here are the most common issues and recommended solutions:

Expression Challenges:

Purification Challenges:

• Multi-domain Nature:

  • The two-domain architecture may lead to flexible conformations affecting chromatographic behavior

  • Solution: Use affinity tags positioned to minimize interdomain interference; consider dual-tagging approaches

• Activity Preservation:

  • Maintaining the coordination between domains during purification is critical for enzymatic function

  • Solution: Include substrate analogs or stabilizing agents in purification buffers; avoid harsh elution conditions

• Protein Aggregation:

  • GMP synthase may form aggregates during concentration steps

  • Solution: Add small amounts of detergent (0.01-0.05% Triton X-100 or 0.005% DDM); maintain glycerol (5-10%) in buffers; concentrate at lower temperatures

Recommended Optimized Protocol:

• Express in yeast expression systems for proper folding and potential glycosylation

• Use affinity chromatography with carefully positioned tags followed by size exclusion chromatography

• Maintain the protein at >85% purity as verified by SDS-PAGE

• Store with 50% glycerol at -20°C/-80°C for long-term stability

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

These strategies help overcome the inherent challenges of working with this complex multi-domain enzyme while preserving its native structure and catalytic function.

How can researchers troubleshoot issues with GMP synthase enzymatic activity assays?

Troubleshooting enzymatic activity assays for GMP synthase requires systematic investigation of multiple factors that can affect catalytic performance:

Common Activity Issues and Solutions:

• Low or No Detectable Activity:

  Potential Causes:

  • Protein denaturation or misfolding

  • Missing cofactors or suboptimal concentrations

  • Inhibitory compounds in the preparation

  Troubleshooting Steps:

  • Verify protein structural integrity via circular dichroism or thermal shift assays

  • Ensure complete reconstitution of lyophilized protein

  • Test with increased enzyme concentrations

  • Add fresh DTT (1-5 mM) to ensure reduced cysteines in the active site

  • Check that all components (ATP, Mg²⁺, XMP, glutamine) are present at appropriate concentrations

• Poor Reproducibility:

  Potential Causes:

  • Enzyme instability

  • Variable substrate quality

  • Inconsistent assay conditions

  Troubleshooting Steps:

  • Prepare fresh working aliquots for each experiment

  • Use single batches of substrates for comparative experiments

  • Control temperature strictly during assays

  • Include internal standards and positive controls

• Inactive GATase Domain:

  Potential Causes:

  • The GATase domain is naturally inactive without allosteric activation

  • Improper domain communication

  Troubleshooting Steps:

  • Pre-incubate the enzyme with ATP·Mg²⁺ and XMP before adding glutamine

  • Verify ammonia-dependent activity (with NH₄Cl instead of glutamine) to confirm ATPPase function

  • Check pH conditions (glutamine-dependent activity optimal at neutral pH)

Optimization Matrix for Activity Assays:

Advanced Troubleshooting Techniques:

• Use mass spectrometry to verify the presence of the adenyl-XMP intermediate

• Apply isothermal titration calorimetry (ITC) to characterize substrate binding

• Perform size exclusion chromatography immediately before assays to eliminate aggregates

By systematically addressing these potential issues, researchers can establish reliable activity assays for recombinant S. maltophilia GMP synthase that provide consistent and biologically relevant results.

How does S. maltophilia GMP synthase compare structurally and functionally to homologous enzymes from other bacterial species?

Comparative analysis of GMP synthases across bacterial species reveals important evolutionary patterns and species-specific adaptations:

Structural Comparison:

Functional Conservation and Divergence:

• Catalytic Mechanism:
  The fundamental chemistry of GMP synthesis is highly conserved across all domains of life, with the reaction proceeding through an adenyl-XMP intermediate . This conservation reflects the essential nature of this metabolic step.

• Substrate Specificity:
  While the core function is conserved, substrate binding pockets may show species-specific adaptations that could be exploited for selective inhibition. These differences are particularly relevant for antimicrobial development.

• Ammonia Channeling:
  The ammonia channel is a conserved feature in all GMP synthases, though the specific residues lining the channel may vary. In bacterial two-domain enzymes like S. maltophilia GMP synthase, the channel is intramolecular, while in archaeal two-subunit enzymes, it forms at the protein-protein interface .

• Regulatory Mechanisms:
  Bacterial GMP synthases typically show tight allosteric coupling between domains, with the GATase domain being inactive without activation by substrate binding to the ATPPase domain . This regulatory feature ensures metabolic efficiency.

Understanding these similarities and differences provides valuable insights for both fundamental research and applied fields such as antimicrobial development, where species-specific features might be targeted for selective inhibition.

What insights can be gained by studying S. maltophilia GMP synthase in the context of microbial pathogenesis and antibiotic resistance?

Studying S. maltophilia GMP synthase offers valuable insights into microbial pathogenesis and potential approaches to overcome antibiotic resistance:

Role in Pathogenesis:

• Metabolic Fitness During Infection:

  • De novo purine biosynthesis is critical for bacterial survival in host environments where nucleotide precursors may be limited

  • GMP synthase activity may be upregulated during certain infection stages to support bacterial replication

• Biofilm Development:

  • S. maltophilia efficiently forms biofilms on biotic and abiotic surfaces, contributing to its antibiotic resistance

  • Guanine nucleotide metabolism influences c-di-GMP levels, which regulate biofilm formation through the Wsp chemosensory system and FleQ transcriptional regulator

  • Mutations affecting guanine nucleotide pools may impact flagellar biosynthesis and cell motility, which play critical roles in S. maltophilia biofilm formation

• Stress Response and Adaptation:

  • Nucleotide metabolism is integrated with bacterial stress responses

  • The ability to modulate GMP synthesis may contribute to S. maltophilia's remarkable adaptability to diverse environments

Connections to Antibiotic Resistance:

• Intrinsic Resistance Mechanisms:

  • S. maltophilia is intrinsically resistant to multiple and broad-spectrum antibiotics

  • Metabolic adaptations, including nucleotide synthesis pathways, may contribute to this intrinsic resistance

• Biofilm-Associated Resistance:

  • Biofilms provide physical barriers against antibiotics

  • Metabolic states within biofilms, potentially influenced by guanine nucleotide availability, can affect antibiotic susceptibility

  • Understanding how GMP synthase activity relates to biofilm formation could reveal new strategies to overcome biofilm-associated resistance

• Target for Anti-Virulence Approaches:

  • Inhibiting GMP synthase may reduce virulence without directly killing bacteria

  • This approach could potentially reduce selective pressure for resistance development

  • Combination therapies targeting both GMP synthase and other pathways could provide synergistic effects

This research direction is particularly valuable given that S. maltophilia infections are increasingly prevalent in healthcare settings and challenging to treat due to the organism's extensive antibiotic resistance profile .

What emerging technologies and methodologies could advance our understanding of GMP synthase function and regulation?

Several cutting-edge technologies and methodologies hold promise for deeper insights into GMP synthase structure, function, and regulation:

Advanced Structural Biology Approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Capture conformational ensembles of GMP synthase in different substrate-bound states

  • Visualize dynamic transitions between inactive and active conformations

  • Resolve the structure of flexible regions often missing in crystal structures

• Integrative Structural Biology:

  • Combine X-ray crystallography, cryo-EM, small-angle X-ray scattering (SAXS), and NMR spectroscopy

  • Develop comprehensive structural models that capture both stable domains and flexible regions

  • Map dynamic protein-protein interactions in multi-enzyme complexes

• Time-Resolved Structural Methods:

  • Use time-resolved X-ray crystallography or time-resolved cryo-EM

  • Capture transient intermediates in the catalytic cycle

  • Visualize the progression of conformational changes during allosteric activation

Novel Functional and Regulatory Insights:

• Single-Molecule Enzymology:

  • Apply single-molecule FRET to observe conformational dynamics in real-time

  • Detect rare catalytic events and conformational states

  • Characterize heterogeneity in enzyme behavior that is masked in ensemble measurements

• Systems Biology Approaches:

  • Integrate transcriptomics, proteomics, and metabolomics to understand GMP synthase in its broader metabolic context

  • Map the dynamic regulation of GMP synthase in response to changing cellular conditions

  • Model the impact of GMP synthase activity on downstream pathways including c-di-GMP signaling and biofilm formation

• In-Cell Studies:

  • Use genetic code expansion to incorporate non-canonical amino acids for in-cell labeling

  • Apply proximity labeling techniques to identify interaction partners in the native cellular environment

  • Develop biosensors to monitor GMP synthase activity in living cells

Emerging Computational Methods:

• Machine Learning for Enzyme Engineering:

  • Predict mutations that enhance catalytic efficiency or alter substrate specificity

  • Identify allosteric networks through statistical analysis of sequence co-evolution

  • Design novel inhibitors targeting species-specific features

• Enhanced Molecular Dynamics:

  • Simulate complete catalytic cycles including ammonia channeling

  • Model allosteric communication pathways between domains

  • Predict effects of mutations on enzyme dynamics and function

These technologies promise to reveal new dimensions of GMP synthase biology with implications for both fundamental understanding and applied research in antimicrobial development.

What potential applications could emerge from in-depth studies of S. maltophilia GMP synthase in biotechnology and medicine?

In-depth studies of S. maltophilia GMP synthase could lead to numerous innovative applications:

Therapeutic Applications:

• Novel Antimicrobial Strategies:

  • Develop selective inhibitors targeting unique features of bacterial GMP synthases

  • Design anti-biofilm agents that disrupt the connection between GMP synthesis and c-di-GMP signaling

  • Create combination therapies targeting both GMP synthase and biofilm formation pathways

• Vaccine Development:

  • Explore recombinant GMP synthase as a potential vaccine antigen

  • Target epitopes unique to S. maltophilia for specific immune responses

  • Develop attenuated bacterial strains with modified GMP synthase for live vaccines

• Diagnostic Tools:

  • Create sensitive detection methods for S. maltophilia based on GMP synthase properties

  • Develop rapid tests for antibiotic susceptibility by monitoring metabolic activities

  • Design biosensors using engineered GMP synthase variants

Biotechnological Applications:

• Biocatalysis and Green Chemistry:

  • Engineer GMP synthase variants capable of synthesizing novel nucleotide analogs

  • Develop environmentally friendly enzymatic routes to pharmaceutically relevant compounds

  • Create immobilized enzyme systems for continuous biocatalytic production

• Synthetic Biology Platforms:

  • Incorporate engineered GMP synthase into synthetic metabolic pathways

  • Develop genetic circuits using GMP synthase-based biosensors

  • Create microbial cell factories with optimized purine metabolism

• Research Tools:

  • Design activity-based probes for studying nucleotide metabolism

  • Develop reporter systems based on GMP synthase allosteric mechanisms

  • Create model systems for studying enzyme channeling and allostery

Biofilm Control Technologies:

• Anti-Biofilm Surfaces:

  • Develop surfaces that release GMP synthase inhibitors to prevent biofilm formation

  • Create materials incorporating enzymes that degrade biofilm components

  • Design smart materials that detect and respond to biofilm formation

• Industrial Applications:

  • Control biofilms in industrial water systems and pipelines

  • Reduce biofouling on marine equipment and vessels

  • Develop cleaning solutions for biofilm removal in food processing equipment

These diverse applications highlight the translational potential of fundamental research on S. maltophilia GMP synthase, spanning from basic enzymatic studies to practical solutions for clinical and industrial challenges.