Recombinant Aliivibrio salmonicida GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the functional role of GMP synthase in Aliivibrio salmonicida?

GMP synthase (GuaA) catalyzes the final step in the de novo GMP biosynthesis pathway, converting XMP to GMP through a two-step reaction. This enzyme first reversibly adenylates XMP to form an O²-adenyl-XMP intermediate using ATP, followed by the amination at the C² position using the amide group from glutamine . This reaction is critical for nucleotide metabolism, which directly impacts bacterial growth and virulence.

In bacterial pathogens like A. salmonicida, GMP synthase activity is essential for survival and pathogenesis. Similar to findings in Clostridioides difficile, where GuaA inactivation caused severe growth defects and reduced infectivity , the GMP synthase in A. salmonicida likely plays a crucial role in its ability to cause cold-water vibriosis by maintaining nucleotide pools necessary for replication within host tissues.

What is the structural organization of A. salmonicida GMP synthase?

Based on structural studies of GMP synthases from other organisms, A. salmonicida GMP synthase likely contains three distinct domains:

• N-terminal ATP pyrophosphatase (ATP-PPase) domain - responsible for the adenylation of XMP

• Glutamine amidotransferase (GATase) domain - catalyzes glutamine hydrolysis to release ammonia

• C-terminal dimerization domain - facilitates formation of functional homodimers

Crystal structures from other bacterial GMP synthases, such as those from E. coli and Thermus thermophilus, confirm that GMP synthase exists as a homodimer . The enzyme requires magnesium as a cofactor for the adenylation reaction, with evidence of cooperative binding observed in homologous enzymes .

What expression systems are optimal for producing recombinant A. salmonicida GMP synthase?

For recombinant expression of A. salmonicida GMP synthase, several considerations are important:

• Host selection: E. coli strains like BL21(DE3) or its derivatives are recommended for high-level expression of bacterial enzymes. For this cold-adapted enzyme from a psychrophilic organism, expression at lower temperatures (15-18°C) may improve proper folding.

• Vector choice: Expression vectors containing T7 promoters with lac operator control (e.g., pET series) allow for regulated expression after IPTG induction.

• Fusion strategy: An N-terminal His-tag facilitates purification while minimizing interference with enzyme function. The tag placement should avoid disrupting the dimerization interface.

• Expression conditions: Using defined media supplemented with trace elements can enhance expression yield. Induction at OD₆₀₀ 0.6-0.8 with 0.1-0.5 mM IPTG, followed by overnight expression at 16°C often produces better results for enzymes from psychrophilic bacteria.

What methods are effective for purifying active recombinant A. salmonicida GMP synthase?

A multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins for His-tagged protein.

• Intermediate purification: Ion exchange chromatography to remove nucleic acid contaminants and impurities.

• Polishing step: Size exclusion chromatography to isolate the properly folded dimeric enzyme and remove aggregates.

Buffer optimization is critical for maintaining activity:

• Include 5-10 mM MgCl₂ to stabilize the enzyme structure

• Add reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation

• Maintain pH between 7.0-8.0 (typically 50 mM Tris-HCl or HEPES)

• Include 10-15% glycerol for storage stability

• Consider adding 100-150 mM NaCl to mimic marine conditions

How do you assess the enzymatic activity of recombinant A. salmonicida GMP synthase?

Multiple complementary approaches can be used to measure GMP synthase activity:

• Spectrophotometric assays:

  • Monitor AMP formation at 290 nm

  • Track pyrophosphate release using coupled enzyme systems

  • Measure NADH oxidation in linked enzyme assays

• Chromatographic methods:

  • HPLC analysis of GMP formation

  • Ion-exchange chromatography to separate nucleotides

• Activity assay conditions:

  • Buffer: 50 mM HEPES or Tris-HCl, pH 7.5-8.0

  • Substrates: 50-200 μM XMP, 1-5 mM ATP, 1-10 mM glutamine

  • Cofactors: 5-10 mM MgCl₂

  • Temperature: 4-25°C (testing across range for cold-adapted enzyme)

• Separate domain activities:

  • GATase activity: measure ammonia production

  • ATP-PPase activity: quantify pyrophosphate release

How does the kinetic profile of A. salmonicida GMP synthase compare to those from other organisms?

While specific kinetic parameters for A. salmonicida GMP synthase are not directly available in the search results, comparison with characterized GMP synthases provides insights:

As a cold-adapted enzyme from a psychrophilic marine bacterium, A. salmonicida GMP synthase would be expected to show higher catalytic efficiency at lower temperatures compared to mesophilic homologs . This adaptation typically involves increased flexibility in substrate binding regions, which often manifests as lower Km values but potentially decreased thermal stability.

What role does GMP synthase play in A. salmonicida virulence and pathogenesis?

Several lines of evidence suggest GMP synthase is crucial for A. salmonicida pathogenesis:

• In C. difficile, inactivation of the riboswitch-controlled GMP synthase (GuaA) led to severe growth defects and poor infectivity in mouse models , suggesting similar importance in A. salmonicida.

• Nucleotide metabolism enzymes are often essential for bacterial adaptation during infection processes. GMP synthase provides GMP needed for:

  • DNA/RNA synthesis during rapid replication

  • Signal molecule production

  • Stress response regulation

• Potential connection to quorum sensing (QS) networks, which are critical for virulence regulation in many bacterial pathogens including Vibrio species . GMP levels may influence:

  • Production of autoinducers

  • Expression of QS-regulated virulence factors

  • Biofilm formation capabilities

Cold adaptation of GMP synthase may be particularly important for A. salmonicida's ability to cause disease in cold-water fish, allowing efficient nucleotide synthesis at low temperatures.

How can structural insights into A. salmonicida GMP synthase inform antimicrobial development?

Understanding the structure-function relationship of A. salmonicida GMP synthase could guide development of selective inhibitors:

• Target-specific binding pockets:

  • ATP-binding site in the ATP-PPase domain

  • Glutamine-binding region in the GATase domain

  • XMP binding pocket

  • Interdomain communication channels

  • Dimerization interface

• Exploiting cold-adaptation features:

  • Regions with increased flexibility

  • Psychrophile-specific surface residues

  • Altered electrostatic interactions

• Selective inhibition strategies:

  • Transition-state analogs targeting the adenylation step

  • Compounds blocking glutamine access or ammonia channeling

  • Allosteric inhibitors affecting communication between domains

  • Molecules disrupting dimerization

• Comparative approaches:

  • Analysis of differences between A. salmonicida and human GMP synthase

  • Focus on bacterial-specific structural elements

Kinetic differences between bacterial and human GMP synthases, such as the significantly higher Km for glutamine in C. neoformans (1130 ± 162 μM) compared to human enzyme (406 ± 49 μM) , suggest potential for selective targeting.

How does riboswitch control of guaA expression function in A. salmonicida?

Based on findings in C. difficile , A. salmonicida guaA expression is likely regulated by a guanine-sensing riboswitch with specific adaptations for marine environments:

• Riboswitch mechanism:

  • Guanine-binding aptamer domain in the 5' UTR

  • Expression regulation through transcription termination or translation initiation control

  • Feedback regulation based on intracellular guanine concentration

• Environmental adaptation:

  • Temperature-responsive elements reflecting psychrophilic lifestyle

  • Potential salt/osmolarity sensing components

  • Integration with marine-specific signaling pathways

• Relationship to virulence:

  • Coordination with quorum sensing networks mentioned in search results

  • Adaptation to host fish environment

  • Regulation during different infection stages

• Experimental approaches:

  • RNA structure probing at different temperatures

  • Reporter gene fusion assays

  • In vitro transcription/translation systems

  • Comparative genomics with other Vibrionaceae

What challenges are associated with studying A. salmonicida GMP synthase structure-function relationships?

Several technical challenges must be addressed:

• Cold adaptation considerations:

  • Maintaining proper folding during expression in mesophilic hosts

  • Preserving activity during purification at room temperature

  • Designing activity assays that account for temperature optima

• Crystallization challenges:

  • Higher structural flexibility may complicate crystal formation

  • Need for specialized crystallization conditions (lower temperatures, marine-mimicking conditions)

  • Potential requirement for ligand-bound forms to stabilize structure

• Function assessment:

  • Distinguishing ATP-PPase and GATase activities

  • Measuring ammonia channeling efficiency

  • Quantifying temperature effects on catalytic parameters

• Comparative analysis:

  • Limited structural data on GMP synthases from psychrophilic organisms

  • Determining which features are cold-adaptation versus marine-adaptation

How can site-directed mutagenesis elucidate the catalytic mechanism of A. salmonicida GMP synthase?

Strategic mutagenesis can provide insights into key functional elements:

• ATP-PPase domain targets:

  • Conserved ATP-binding residues

  • XMP interaction sites

  • Mg²⁺ coordination residues

• GATase domain targets:

  • Catalytic triad involved in glutamine hydrolysis

  • Ammonia channeling residues

  • Substrate specificity determinants

• Interdomain interfaces:

  • Residues mediating domain communication

  • Ammonia channel components

  • Conformational change facilitators

• Cold adaptation features:

  • Surface-exposed flexible loops

  • Salt bridges and electrostatic interactions

  • Hydrophobic core residues

• Experimental approaches:

  • Alanine scanning of conserved motifs

  • Conservative substitutions to probe specific interactions

  • Creation of chimeric enzymes with mesophilic homologs

  • Introduction of thermostabilizing mutations

Understanding the dual catalytic action required for amination of XMP to GMP involves analyzing both the initial adenyl-XMP formation and subsequent reaction with glutamine-derived ammonia . Mutations affecting either process would provide valuable mechanistic insights.

What is the relationship between A. salmonicida GMP synthase and quorum sensing systems?

A potential link exists between GMP synthase activity and quorum sensing (QS) networks:

• Nucleotide signaling connection:

  • Purine nucleotides may influence secondary messenger systems

  • GMP-derived signals could affect virulence regulation

• Evidence from related systems:

  • Quorum sensing/quenching affects virulence in many bacterial pathogens

  • Vibrio species utilize QS for controlling virulence factor expression

  • "Luminescent vibriosis" in shrimp involves QS-controlled phenotypes

• Potential mechanisms:

  • Guanine nucleotide availability affecting signal molecule synthesis

  • Regulatory overlap between nucleotide metabolism and QS circuits

  • Shared transcriptional control mechanisms

• Experimental approaches:

  • Analyzing QS-regulated phenotypes in GuaA-depleted strains

  • Measuring autoinducer production under varying guanine availability

  • Transcriptomic analysis comparing wild-type and guaA mutants