Recombinant Escherichia coli O45:K1 GMP synthase [glutamine-hydrolyzing] (guaA)

What is GMP synthase (guaA) and its function in E. coli metabolism?

GMP synthase (EC 6.3.4.1), encoded by the guaA gene, functions as a glutamine amidotransferase that catalyzes the final step in de novo GMP biosynthesis. The enzyme specifically catalyzes the conversion of xanthosine monophosphate (XMP) to guanosine monophosphate (GMP), using glutamine as the amino group donor . This reaction is critical for purine nucleotide biosynthesis in E. coli, particularly when the organism cannot salvage guanine from the environment.

The enzyme contains two distinct catalytic activities operating in separate domains:

• Glutaminase domain: Hydrolyzes glutamine to produce ammonia

• Synthetase domain: Transfers the ammonia to XMP, forming GMP

This dual-domain structure enables the coordinated transfer of ammonia between catalytic sites via an internal channel that protects the reactive ammonia from the cellular environment .

What is the genetic organization of the guaA gene in E. coli?

The guaA gene is part of the polycistronic guaBA operon in E. coli. The structural gene encodes a protein of 525 amino acid residues with a calculated molecular weight of 58,604 Da . The guaA gene is separated from the upstream guaB gene (which encodes IMP dehydrogenase) by a 68-base pair intercistronic region .

The operon organization is significant for coordinated regulation, as both enzymes (GuaA and GuaB) function sequentially in the pathway from IMP to GMP. The 3' end of the guaA mRNA is located 36-37 nucleotides downstream of the translation stop codon within a region of dyad symmetry that resembles a rho-independent transcription termination site .

What is the role of the K1 capsule in E. coli and how might it relate to protein expression?

The K1 capsule is a homopolymer of α−2,8-linked N-acetylneuraminic acid (sialic acid; NeuNAc) termed polysialic acid (polySia) that mimics human neuronal and immune cell modifications . This molecular mimicry has significant implications for bacterial virulence and host interactions.

Key points about the K1 capsule:

• Present in approximately 25% of E. coli blood stream infection isolates

• Confers resistance to complement- and phagocyte-mediated killing

• Enhances bacterial survival in human serum independent of genetic background

• Associated with invasive infections, particularly bloodstream infections and neonatal meningitis

• Emerged in multiple E. coli lineages over the past 500 years

How is the guaA gene regulated in E. coli?

The guaA gene in E. coli is subject to transcriptional regulation as part of the guaBA operon. Key aspects of this regulation include:

This regulatory mechanism ensures that E. coli allocates metabolic resources efficiently, synthesizing GMP de novo only when it cannot be obtained through less energy-intensive salvage pathways.

What are the key structural characteristics of E. coli GMP synthase?

E. coli GMP synthase exhibits a complex structure reflecting its bifunctional nature:

• Two-domain architecture: The enzyme consists of:

  • An N-terminal glutaminase domain with the glutamine binding site

  • A C-terminal synthetase domain containing the XMP binding site

• Ammonia channel: A key structural feature is the intramolecular tunnel connecting the two active sites, allowing nascent ammonia to travel from the glutaminase domain to the synthetase domain without exposure to solvent .

• Metal binding site: The enzyme contains zinc, with approximately 1 mol of zinc per mol of subunit, which plays a role in catalysis .

• Conformational changes: The enzyme undergoes significant conformational changes during catalysis, which facilitate the coordination between the two catalytic domains and the opening/closing of the ammonia channel .

• Conservation: Despite detailed structural analysis, some aspects of inter-domain crosstalk in GMPS remained unclear until relatively recent studies, despite the first structure being published in 1996 .

What are the optimal conditions for expression of recombinant E. coli O45:K1 GMP synthase?

The optimal expression of recombinant E. coli GMP synthase depends on several experimental parameters:

Media composition:

• Defined media offer better reproducibility and are preferred for structural studies

• Complex media typically yield higher protein amounts but with greater batch-to-batch variation

• Autoinduction methods can be particularly effective for GMP synthase expression

Expression system parameters:

• Temperature: 25-30°C often provides a balance between expression level and solubility

• Induction: Gradual induction (autoinduction) or low IPTG concentrations (0.1-0.5 mM) typically yield better results than strong induction

• Growth phase: Induction at mid-log phase (OD600 0.6-0.8) is generally optimal

• Duration: 4-6 hours post-induction at 37°C or overnight at lower temperatures

Expression vector considerations:

• Vectors with the pMAL-c2 system have been successfully used for expression of similar enzymes in E. coli

• N-terminal fusion tags (His, MBP) can improve solubility while maintaining enzyme activity

• Codon optimization may improve expression, especially for rare codons

The metabolic impact of overexpression should be considered, as high-level production of recombinant proteins can significantly alter the metabolic balance in E. coli, potentially affecting growth rates and final yields .

How does the catalytic mechanism of GMP synthase work across its two domains?

The catalytic mechanism of GMP synthase involves coordinated reactions across its two domains:

Glutaminase Domain Reaction:

• Binding of glutamine to the N-terminal domain

• Nucleophilic attack by a catalytic cysteine residue on the γ-amide carbon of glutamine

• Formation of a tetrahedral intermediate

• Collapse of the intermediate, releasing ammonia (NH3)

• Hydrolysis of the acyl-enzyme intermediate, releasing glutamate

Synthetase Domain Reaction:

• Binding of XMP and ATP to the C-terminal domain

• ATP-dependent activation of XMP, forming an adenylated XMP intermediate

• Channel-directed transport of ammonia from the glutaminase domain

• Nucleophilic attack by ammonia on the activated C2 position of XMP

• Release of AMP and inorganic phosphate

• Formation of GMP

The coordination between these domains involves:

• Conformational changes triggered by substrate binding

• Opening of the ammonia channel only when both domains have bound their respective substrates

• Allosteric communication ensuring that glutamine hydrolysis occurs only when XMP is ready for amination

This intricate mechanism ensures efficient nitrogen transfer while preventing wasteful glutamine hydrolysis in the absence of XMP substrate .

What methodological approaches are recommended for purification of recombinant GMP synthase?

Purification of recombinant GMP synthase from E. coli O45:K1 requires a strategic approach:

Initial extraction and clarification:

• Cell lysis: Sonication or pressure-based methods in a buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 1 mM DTT

• Removal of cellular debris by centrifugation (20,000 × g, 30 min, 4°C)

• Filtration of supernatant through a 0.45 μm filter

Chromatographic purification sequence:

• Affinity chromatography:

  • For His-tagged constructs: Ni-NTA chromatography with step gradient elution

  • For MBP-tagged constructs: Amylose resin with maltose elution

• Ion exchange chromatography:

  • Anion exchange (Q Sepharose) at pH 7.5-8.0 with a 0.1-1 M NaCl gradient

• Size exclusion chromatography:

  • Superdex 200 in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT

Tag removal considerations:

• TEV or Factor Xa protease digestion for tag removal

• Second affinity step to remove uncleaved protein and free tag

Storage conditions:

• 50% glycerol at -20°C or flash-frozen aliquots at -80°C

• Addition of 5-10% glycerol and 1 mM DTT to prevent activity loss

Typical yield from a 1-liter culture is 10-15 mg of purified enzyme, with specific activity of approximately 3.2 s−1 with guanine as substrate (for related enzymes) .

How can site-directed mutagenesis be used to study GMP synthase function?

Site-directed mutagenesis provides powerful insights into GMP synthase structure-function relationships:

Key residues for targeted mutagenesis:

• Catalytic residues:

  • Glutaminase domain: The conserved catalytic triad (Cys-His-Glu)

  • Synthetase domain: ATP-binding residues and XMP-interacting residues

• Channel-forming residues:

  • Residues lining the ammonia channel between domains

  • Gating residues that control channel opening/closing

• Metal-binding residues:

  • The nine-residue heavy metal binding site (PG[FL]VDTHIH) shared with human guanine deaminase

  • Coordination sphere residues for zinc binding

Mutagenesis protocol outline:

• Design mutagenic primers containing the desired mutation flanked by 15-20 nucleotides of correct sequence

• Perform PCR using a high-fidelity polymerase (e.g., Pfu or Vent)

• Digest template DNA with DpnI

• Transform into competent E. coli

• Screen colonies by sequencing

• Express and purify mutant proteins

Functional analysis of mutants:

• Enzyme kinetics to determine effects on Km and kcat

• Thermal stability measurements to assess structural impacts

• Domain interaction studies using chemical crosslinking or FRET

• Substrate binding assays to identify roles in substrate recognition

This approach has revealed that mutations in the ammonia channel can decouple the two catalytic activities, while mutations at the domain interface can affect allosteric communication between the domains .

What methods are available for assessing GMP synthase activity in vitro?

Several complementary methods can be employed to measure GMP synthase activity:

Spectrophotometric assays:

• Coupled enzyme assay:

  • GMP production coupled to GMP reductase and NADPH oxidation

  • Monitored at 340 nm for NADPH consumption

  • Reaction mixture: 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.2 mM NADPH, 2 mM ATP, 0.5 mM XMP, 5 mM glutamine, GMP reductase

• Direct measurement of glutamate production:

  • Glutamate dehydrogenase coupled assay with NAD+ reduction

  • Monitored at 340 nm for NADH formation

Chromatographic methods:

• HPLC analysis:

  • C18 reverse-phase column

  • Mobile phase: 50 mM potassium phosphate pH 6.0, 5% methanol

  • UV detection at 254 nm

  • Allows direct quantification of XMP consumption and GMP formation

• Thin-layer chromatography:

  • PEI-cellulose plates

  • Development in 0.75 M LiCl

  • Visualization under UV light

Isotopic methods:

• Radioactive substrate incorporation:

  • [14C]-glutamine or [15N]-glutamine as substrate

  • Measurement of labeled GMP formation

Activity calculation:
The specific activity is typically expressed as micromoles of GMP formed per minute per milligram of protein, with reported values for E. coli GMP synthase of approximately 3.2 s−1 with guanine as substrate .

How does the K1 capsule affect research with recombinant proteins in E. coli O45:K1?

Working with E. coli O45:K1 for recombinant protein expression presents unique considerations due to the K1 capsule:

Impact on cell physiology:
The K1 capsule enhances E. coli survival in human serum independent of genetic background and affects bacterial interactions with the environment . This can influence:

• Cell membrane properties

• Protein secretion and transport

• Cell aggregation behavior

• Biofilm formation

Experimental considerations:

• Safety precautions:

  • The K1 capsule is associated with virulence and invasive infections

  • Enhanced laboratory safety protocols may be necessary

  • BSL-2 practices should be implemented

• Extraction efficiency:

  • The polysaccharide capsule may impact cell lysis efficiency

  • Modified lysis buffers may be required

  • Enzymatic pre-treatment with capsule-degrading enzymes can improve protein extraction

• Purification challenges:

  • Capsular material can contaminate protein preparations

  • Additional purification steps may be necessary

  • Nuclease and glycosidase treatments may improve purity

• Genetic stability:

  • The K1 capsule locus (K1-cps) spans multiple genes

  • Genetic stability of the recombinant system should be monitored

  • Expression plasmid compatibility with the K1-cps must be considered

The choice of E. coli O45:K1 as an expression host should be carefully evaluated against these factors, with particular attention to the potential virulence of this strain due to the K1 capsule association with severe infections .

What structural changes occur during the catalytic cycle of GMP synthase?

The catalytic cycle of GMP synthase involves significant conformational changes:

Apo-enzyme structure:

• Open conformation with separated domains

• Inactive glutaminase domain

• Accessible synthetase active site

• Closed ammonia channel

Upon substrate binding:

• ATP and XMP binding to the synthetase domain triggers:

  • Conformational change in the synthetase domain

  • Domain closure around the substrates

  • Partial opening of the ammonia channel

  • Signal transmission to the glutaminase domain

• Glutamine binding to the glutaminase domain leads to:

  • Loop movements around the glutamine binding site

  • Activation of the catalytic triad

  • Complete opening of the ammonia channel

  • Formation of a fully functional enzyme complex

During catalysis:

• Ammonia traverses the hydrophobic channel

• Further conformational adjustments coordinate the two reactions

• Glutamate release from the glutaminase domain

• GMP formation in the synthetase domain

Product release:

• Domain reopening

• Channel closure

• Return to the apo-enzyme conformation

These orchestrated structural changes ensure that the two catalytic activities are tightly coupled, preventing wasteful glutamine hydrolysis when XMP is not available for amination .

How can isotope labeling be used to study nitrogen transfer in GMP synthase?

Isotope labeling provides powerful insights into the ammonia transfer mechanism in GMP synthase:

15N-glutamine incorporation experiments:

• Experimental protocol:

  • Incubate GMP synthase with [15N]-labeled glutamine, ATP, and XMP

  • Isolate GMP product

  • Analyze by mass spectrometry or NMR

• Key parameters:

  • Reaction buffer: 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT

  • Enzyme concentration: 0.1-0.5 μM

  • Substrate concentrations: 5 mM [15N]-glutamine, 2 mM ATP, 0.5 mM XMP

  • Incubation time: 30-60 minutes at 37°C

• Data interpretation:

  • Transfer of 15N from glutamine to GMP confirms intramolecular channeling

  • Quantification of 15N incorporation efficiency

  • Analysis of nitrogen position in GMP structure

Kinetic isotope effect studies:

• Comparison of reaction rates with 14N and 15N glutamine

• Determination of primary and secondary isotope effects

• Mechanistic insights into rate-limiting steps

Channel investigation experiments:

• Heavy-isotope labeling of channel-lining residues

• Mass spectrometry analysis of hydrogen-deuterium exchange

• Correlation of channel dynamics with catalytic rate

These approaches have demonstrated that the ammonia produced by glutamine hydrolysis is efficiently channeled to the synthetase domain with minimal loss to the surrounding medium, confirming the functional importance of the intramolecular tunnel in GMP synthase .

What are the comparative differences between E. coli GMP synthase and GMP synthase from other organisms?

Comparative analysis of GMP synthase across species reveals important evolutionary and functional insights:

Structural organization comparison:

Functional differences:

• Catalytic efficiency:

  • E. coli GMP synthase: Km ≈ 15 μM for guanine, kcat ≈ 3.2 s-1

  • Human GMP synthase: Generally lower catalytic efficiency

  • P. falciparum GMP synthase: Higher affinity for glutamine

• Regulatory mechanisms:

  • E. coli: Regulated primarily through the PurR repressor system

  • Eukaryotes: Often subject to complex allosteric regulation

  • Human: Additional post-translational modifications

• Inhibitor sensitivity:

  • E. coli GMP synthase: More sensitive to certain antibiotics

  • Human enzyme: Distinct inhibitor profile

  • Parasite enzymes: Unique sensitivities exploitable for therapeutics

Evolutionary insights:

• Core catalytic residues are highly conserved across all domains of life

• Channel-forming residues show more variation between prokaryotes and eukaryotes

• Metal-binding motifs are conserved but with organism-specific variations

These differences provide opportunities for:

• Development of selective inhibitors for antimicrobial applications

• Understanding evolutionary constraints on enzyme function

• Rational design of GMP synthase variants with altered properties

How do changes in metabolic balance impact GMP synthase expression and function in E. coli?

The expression and function of GMP synthase in E. coli are intimately connected to cellular metabolic balance:

Transcriptional regulation responses:

• Purine availability:

  • High guanine levels repress guaA expression via PurR

  • Reduced purine levels induce expression

  • GuaB (IMP dehydrogenase) and GuaA are co-regulated in response to purine levels

• Nutrient limitation effects:

  • Carbon source limitation alters guaA expression

  • Nitrogen limitation increases glutamine conservation pathways

  • Amino acid starvation triggers stringent response affecting guaA

Metabolic integration:

Recombinant protein production impact:
High-level recombinant protein production creates significant metabolic burden that affects GMP synthase through:

• Redirection of cellular resources

• Amino acid depletion

• Altered energy balance

• Stress response activation

What are common obstacles in purifying active recombinant GMP synthase and their solutions?

Researchers frequently encounter challenges when purifying functional GMP synthase from recombinant E. coli systems:

Challenge 1: Poor solubility

• Symptoms: Majority of protein in inclusion bodies, low yield in soluble fraction

• Solutions:

  • Lower induction temperature (16-25°C)

  • Reduce inducer concentration

  • Use solubility-enhancing tags (MBP, SUMO)

  • Co-express chaperones (GroEL/GroES, DnaK/DnaJ)

  • Add solubility enhancers to lysis buffer (0.1% Triton X-100, 5-10% glycerol)

Challenge 2: Low enzymatic activity

• Symptoms: Protein appears pure but shows reduced or no activity

• Solutions:

  • Maintain reducing conditions (1-5 mM DTT or 0.5-2 mM β-mercaptoethanol)

  • Add zinc (10-50 μM ZnCl2) to purification buffers

  • Include ATP and Mg2+ (1 mM each) in storage buffer

  • Avoid freeze-thaw cycles

  • Purify at 4°C throughout the procedure

Challenge 3: Proteolytic degradation

• Symptoms: Multiple bands on SDS-PAGE, decreasing yield during purification

• Solutions:

  • Add protease inhibitors (PMSF, EDTA-free cocktail)

  • Reduce purification time

  • Maintain low temperature

  • Use protease-deficient host strains

  • Design constructs with improved stability

Challenge 4: Copurifying contaminants

• Symptoms: Persistent impurities, activity interference

• Solutions:

  • Add nuclease treatment (Benzonase) to remove nucleic acids

  • Include high salt wash steps (0.5-1 M NaCl)

  • Add ATP wash (5 mM) to remove chaperones

  • Use orthogonal purification methods

  • Consider on-column refolding protocols

These troubleshooting strategies should be implemented systematically, with activity assays at each step to track enzyme functionality throughout the purification process.

How can researchers resolve inconsistent activity results with recombinant GMP synthase?

When encountering variable or inconsistent activity measurements with GMP synthase, consider the following methodological approaches:

Source of variability diagnosis:

• Enzyme stability assessment:

  • Monitor activity over time under storage conditions

  • Test different buffer compositions for stability enhancement

  • Determine half-life at different temperatures

• Assay component verification:

  • Use fresh ATP and glutamine preparations

  • Verify XMP purity and concentration

  • Check Mg2+ concentration (5-10 mM optimal)

  • Ensure adequate reducing agent concentration

• Metal content analysis:

  • Measure zinc content using atomic absorption spectroscopy

  • Validate ~1 mol zinc per mol enzyme subunit

  • Test activity response to zinc supplementation

Standardization protocol:

• Reference standard development:

  • Create stable enzyme standard aliquots

  • Use internal controls across experiments

  • Develop activity normalization procedures

• Robust assay conditions:

  • Buffer: 50 mM HEPES pH 7.5 instead of Tris to avoid temperature sensitivity

  • Include 10% glycerol for stability

  • Pre-incubate enzyme with Mg2+ before substrate addition

  • Control temperature precisely (±0.5°C)

• Data analysis refinement:

  • Calculate initial rates only from linear portion of progress curves

  • Use replicate measurements (minimum triplicate)

  • Apply statistical outlier tests

  • Consider enzyme-specific normalization factors

By implementing these approaches systematically, researchers can significantly reduce variability in GMP synthase activity measurements and improve experimental reproducibility.

What emerging approaches could advance our understanding of E. coli GMP synthase structure and function?

Several cutting-edge methodologies show promise for deepening our understanding of GMP synthase:

Cryo-Electron Microscopy (Cryo-EM):

• Application to capture conformational states during catalysis

• Potential to visualize domain movements at near-atomic resolution

• Advantage of requiring smaller sample quantities than crystallography

• Opportunity to observe transient catalytic intermediates

Time-Resolved Structural Methods:

• X-ray free electron laser (XFEL) studies to capture rapid structural changes

• Time-resolved crystallography using triggered reactions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein dynamics

Computational Approaches:

• Molecular dynamics simulations of ammonia transfer through the channel

• Quantum mechanics/molecular mechanics (QM/MM) studies of catalytic mechanisms

• Machine learning predictions of allosteric networks within the enzyme

Single-Molecule Biophysics:

• FRET-based approaches to monitor domain movements in real-time

• Optical tweezers to measure forces involved in conformational changes

• Single-molecule enzymology to detect reaction intermediates

Systems Biology Integration:

• Multi-omics approaches to understand GMP synthase in metabolic networks

• Flux analysis to quantify the impact of GMP synthase on purine metabolism

• Genome-scale models incorporating GMP synthase regulation

These approaches will likely reveal new insights into how the enzyme's structure facilitates ammonia channeling, the precise timing of conformational changes during catalysis, and the integration of GMP synthase into the broader cellular metabolic network.

What potential biotechnological applications exist for recombinant E. coli GMP synthase?

GMP synthase offers several promising biotechnological applications:

Biocatalytic Applications:

• Enzymatic synthesis of modified guanosine nucleotides for pharmaceutical applications

• Production of labeled nucleotides for structural biology research

• Development of biosensors for glutamine or GMP detection

• Green chemistry approaches to nucleotide synthesis

Therapeutic Target Development:

• Structure-based design of antibiotics targeting bacterial GMP synthase

• Screening platforms for selective inhibitors against pathogenic E. coli strains

• Comparative studies with human GMP synthase for selectivity optimization

• Development of attenuated vaccine strains through guaA modification

Metabolic Engineering Applications:

• Enhancement of nucleotide production in biotechnology strains

• Modulation of GMP levels for improved recombinant protein yields

• Integration into synthetic biology circuits for metabolic control

• Development of auxotrophic selection systems based on guaA

Diagnostic Tools:

• Development of activity-based probes for bacterial detection

• Design of reporter systems based on GMP synthase regulation

• Immunodiagnostic applications using recombinant enzyme as antigen

• Point-of-care nucleotide detection systems