Recombinant Edwardsiella ictaluri GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is GMP synthase [glutamine-hydrolyzing] (guaA) in Edwardsiella ictaluri?

GMP synthase [glutamine-hydrolyzing] (guaA) is an enzyme encoded by the guaA gene in Edwardsiella ictaluri, a gram-negative fish pathogen that causes enteric septicemia in catfish . This enzyme belongs to the glutamine amidotransferase family, which catalyzes the amination of various molecules using the amide nitrogen from glutamine . Specifically, GMP synthase catalyzes the final step in GMP biosynthesis, converting XMP (xanthosine monophosphate) to GMP (guanosine monophosphate) using ATP and glutamine as substrates. The enzyme demonstrates complex multi-active-site regulation and interdomain communication typical of glutamine amidotransferases .

How does E. ictaluri GMP synthase compare structurally to other bacterial GMP synthases?

E. ictaluri GMP synthase likely shares structural features with other bacterial GMP synthases, particularly those from E. coli which have been more extensively studied. The enzyme contains multiple tryptophan residues dispersed throughout its structure that are critical for monitoring conformational changes . Like other GMP synthases, it likely possesses two main functional domains: a glutaminase domain that hydrolyzes glutamine to generate ammonia, and a synthetase domain that uses this ammonia to aminate XMP, forming GMP. The study of GMP synthase from other organisms has shown that it undergoes significant conformational changes upon binding of its nucleotide substrates, suggesting a complex tertiary structure with dynamic properties essential for its catalytic function .

What is the role of E. ictaluri GMP synthase in bacterial metabolism and pathogenesis?

GMP synthase plays a central role in purine nucleotide biosynthesis, which is essential for the growth and survival of E. ictaluri. While not specifically identified among the documented virulence genes (citC, gadB, katB, mukF, and fimA) , GMP synthase likely contributes indirectly to pathogenicity by enabling bacterial replication within host tissues. As E. ictaluri is described as a "nasal/oral invasive intracellular pathogen" , its ability to proliferate inside host cells would depend on robust nucleotide synthesis pathways. The essential nature of guaA makes it a potential target for vaccine development strategies, similar to the balanced-lethal system developed with the asdA gene in E. ictaluri for recombinant attenuated Edwardsiella vaccines (RAEV) .

What expression systems are optimal for recombinant E. ictaluri GMP synthase production?

Recombinant E. ictaluri GMP synthase can be expressed in multiple host systems, each offering distinct advantages for different research purposes:

According to product specifications, recombinant preparations can achieve "greater or equal to 85% purity as determined by SDS-PAGE" regardless of the expression system used .

What analytical methods can effectively characterize the conformational dynamics of E. ictaluri GMP synthase?

Stopped-flow tryptophan fluorescence spectroscopy provides valuable insights into the conformational dynamics of GMP synthase. This technique reveals that the enzyme undergoes significant conformational changes upon binding its nucleotide substrates . When GMP synthase (premixed with ATP) is combined with XMP substrate, a decay in intrinsic tryptophan fluorescence occurs, indicating alteration in the environment of one or more of the enzyme's tryptophan residues . The conformational change appears sequential, with an initial lag phase (<10ms) that decreases with increasing substrate concentration, followed by a decay phase representing the actual conformational adjustment . This methodology could be applied to studying E. ictaluri GMP synthase by:

• Monitoring intrinsic tryptophan fluorescence changes upon substrate binding

• Creating tryptophan-to-phenylalanine mutants to identify specific residues involved in conformational changes

• Analyzing the kinetics of these changes to determine rate constants for binding and conformational change steps

How can the adenylylated XMP intermediate in the GMP synthase reaction be isolated and characterized?

The adenylylated XMP intermediate formed during the GMP synthase reaction can be isolated and characterized using several approaches:

• HPLC separation: Mixing GMP synthase with ATP and XMP substrates (without glutamine) leads to the formation of a distinct peak in HPLC analysis corresponding to the adenylylated XMP intermediate .

• Verification through substrate addition: The identity of this intermediate can be confirmed by adding glutamine to the reaction mixture, which causes the intermediate peak to disappear concomitant with the appearance of the GMP product peak .

• Scale-up preparation: Large-scale enzyme preparation facilitates isolation of sufficient intermediate for detailed spectral analysis .

• Stability analysis: The enzyme-bound adenylylated XMP intermediate is stable enough to be separated and analyzed, making it accessible for characterization studies .

This intermediate represents a crucial step in understanding the catalytic mechanism and conformational dynamics of GMP synthase.

How can site-directed mutagenesis be applied to study E. ictaluri GMP synthase function?

Site-directed mutagenesis provides a powerful approach for investigating structure-function relationships in E. ictaluri GMP synthase. Based on experimental methods described for other GMP synthases, researchers can implement the following protocol:

• Tryptophan-to-phenylalanine mutations:

  • Use the QIAGEN Quickchange site-directed mutagenesis method

  • Design two complementary "mutagenizing" primers containing the desired mutation

  • Include silent mutations that add or remove restriction sites for easy confirmation

  • Use a pET vector harboring His-tagged GMP synthase as template

• Target selection strategies:

  • Identify conserved residues through sequence alignment with characterized GMP synthases

  • Focus on residues in predicted catalytic sites or domain interfaces

  • Create systematic mutations of tryptophan residues to map conformational changes

  • Design mutations to probe substrate specificity and catalytic efficiency

• Functional analysis of mutants:

  • Measure enzymatic activity using HPLC detection of GMP formation

  • Analyze fluorescence changes upon substrate binding

  • Determine the impact on intermediate formation and stability

This approach allows systematic mapping of residues critical for substrate binding, catalysis, and interdomain communication.

What methods can be used to study the interdomain communication in E. ictaluri GMP synthase?

Interdomain communication in GMP synthase is crucial for coordinating the glutaminase and synthetase activities. Several experimental approaches can investigate this phenomenon:

• Domain-specific mutations:

  • Introduce mutations at predicted domain interfaces

  • Create chimeric enzymes with domains from different species

  • Analyze the impact on catalytic activity and conformational changes

• Biophysical characterization:

  • Stopped-flow fluorescence spectroscopy to detect conformational changes

  • FRET (Förster Resonance Energy Transfer) to measure domain movements

  • Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• Structural analysis:

  • X-ray crystallography or cryo-EM to capture different conformational states

  • Compare structures with and without bound substrates or intermediates

  • Molecular dynamics simulations to model domain movements and communication pathways

These approaches can reveal how substrate binding in one domain triggers conformational changes that activate the other domain, a critical aspect of GMP synthase's catalytic mechanism.

How can researchers assess the impact of environmental conditions on E. ictaluri GMP synthase activity?

Assessment of environmental factors on GMP synthase activity is particularly relevant given E. ictaluri's role as a fish pathogen. A comprehensive experimental design would include:

• Temperature-dependent activity:

  • Measure enzyme activity across a range of temperatures relevant to fish hosts

  • Determine temperature optima and compare with the typical temperature range of catfish environments

  • Analyze thermal stability through differential scanning fluorimetry

• pH dependence:

  • Evaluate activity across pH ranges encountered during infection (gut, tissue, intracellular environments)

  • Measure conformational changes at different pH values using tryptophan fluorescence

  • Assess stability and folding at various pH conditions

• Ion effects:

  • Determine the impact of varying Mg²⁺ concentrations (required for ATP binding)

  • Investigate effects of other physiologically relevant ions (K⁺, Na⁺, Ca²⁺)

  • Analyze salt tolerance relevant to freshwater versus brackish environments

• Oxidative stress response:

  • Measure activity in the presence of reactive oxygen species encountered during host immune response

  • Identify potential redox-sensitive residues through targeted mutagenesis

  • Evaluate protective mechanisms against oxidative inactivation

How can E. ictaluri GMP synthase be targeted for vaccine development strategies?

E. ictaluri GMP synthase could be leveraged for vaccine development through several strategic approaches:

• Balanced-lethal complementation system:

  • Create a guaA deletion mutant (ΔguaA) with impaired growth capability

  • Complement with a plasmid expressing functional guaA

  • Establish plasmid dependence similar to the asdA-based system described for E. ictaluri

• Attenuated vaccine strain development:

  • Engineer conditional guaA expression to create attenuated strains

  • Evaluate attenuation in fish challenge models similar to those used for E. anguillarum virulence testing in milkfish

  • Optimize for bath/oral delivery, leveraging E. ictaluri's natural route of infection

• Antigen delivery platform:

  • Use the guaA-based balanced-lethal system to express heterologous protective antigens

  • Similar to how "Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids"

  • Design multivalent vaccines expressing antigens from various fish pathogens

This approach builds upon the successful balanced-lethal system already demonstrated in E. ictaluri, which represents "the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry" .

What comparative analyses can reveal about the evolution of GMP synthase in Edwardsiella species?

Comparative analysis of GMP synthase across Edwardsiella species could provide valuable insights into evolutionary relationships and functional adaptations:

• Phylogenetic analysis:

  • Compare guaA sequences across Edwardsiella species (E. tarda, E. piscicida, E. anguillarum, E. ictaluri)

  • Correlate with the phylogenetic relationships established through gyrB gene analysis

  • Identify clade-specific sequence variations that might reflect host adaptation

• Structure-function relationships:

  • Map sequence variations onto structural models to identify potentially functionally significant differences

  • Correlate sequence variations with differences in host range or virulence

  • Compare with GMP synthases from other bacterial pathogens to identify convergent evolution

• Selective pressure analysis:

  • Calculate dN/dS ratios to identify regions under positive or purifying selection

  • Correlate with host shifts or environmental adaptations

  • Compare with other metabolic genes to determine if guaA is evolving at similar rates

How can structural studies of E. ictaluri GMP synthase inform inhibitor design for therapeutic applications?

Structural insights into E. ictaluri GMP synthase can guide rational inhibitor design for potential therapeutic applications:

• Active site mapping:

  • Identify catalytic residues through site-directed mutagenesis and activity assays

  • Characterize the adenylylated XMP intermediate binding pocket

  • Model substrate binding interactions to identify critical contact points

• Transition state analysis:

  • Determine the structure of transition state analogs bound to the enzyme

  • Identify stabilizing interactions that could be exploited for inhibitor design

  • Develop transition state mimics as potential high-affinity inhibitors

• Allosteric site identification:

  • Map conformational changes upon substrate binding

  • Identify potential allosteric sites that could be targeted to prevent necessary conformational changes

  • Design inhibitors that lock the enzyme in inactive conformations

• Species-specific targeting:

  • Compare E. ictaluri GMP synthase with host (fish) GMP synthase

  • Identify structural differences that could be exploited for selective inhibition

  • Design inhibitors that preferentially target bacterial over host enzyme

These approaches could lead to novel therapeutics for controlling enteric septicemia in catfish aquaculture, addressing a significant economic challenge in the industry.

What CRISPR-Cas9 strategies can facilitate E. ictaluri GMP synthase research?

CRISPR-Cas9 genome editing offers powerful approaches for studying E. ictaluri GMP synthase:

• Precise gene modifications:

  • Create clean knockouts of guaA to study essentiality and phenotypic consequences

  • Introduce point mutations to study specific amino acid functions in vivo

  • Tag the endogenous enzyme with reporters for localization studies

• Regulatable expression systems:

  • Engineer inducible/repressible guaA expression to study dose-dependent effects

  • Create conditional lethal strains for vaccine development

  • Implement CRISPRi for partial knockdown to identify threshold activity requirements

• High-throughput functional genomics:

  • Create CRISPR libraries targeting guaA regulatory elements

  • Screen for synthetic lethality with other metabolic genes

  • Identify genetic interactions through combinatorial targeting

These approaches would complement traditional methods and could accelerate both basic research and applied vaccine development for E. ictaluri.

How might synthetic biology approaches enhance applications of E. ictaluri GMP synthase?

Synthetic biology offers innovative strategies for harnessing E. ictaluri GMP synthase:

• Engineered enzyme variants:

  • Design synthetic GMP synthases with enhanced catalytic efficiency

  • Create thermostable variants for industrial applications

  • Develop substrate specificity alterations for novel nucleotide analog production

• Modular vaccine platforms:

  • Construct standardized balanced-lethal expression cassettes based on guaA

  • Develop plug-and-play antigen expression systems for rapid vaccine prototyping

  • Engineer strains with programmable attenuation profiles

• Biosensor development:

  • Create GMP synthase-based biosensors for detecting nucleotide precursors

  • Develop reporter systems for monitoring bacterial metabolism in vivo

  • Design diagnostic tools for monitoring E. ictaluri infection in aquaculture

These synthetic biology applications could transform both research tools and practical applications in aquaculture disease management.

What integrative approaches can connect GMP synthase function to broader bacterial metabolic networks?

Understanding GMP synthase within the context of broader bacterial metabolism requires integrative approaches:

• Systems biology analysis:

  • Map metabolic flux through purine biosynthesis pathways

  • Identify regulatory connections between nucleotide metabolism and other pathways

  • Model the impact of environmental changes on metabolic network functioning

• Multi-omics integration:

  • Correlate transcriptomic data on guaA expression with metabolomic profiles

  • Link proteomic analysis of GMP synthase abundance with pathway activity

  • Identify post-translational modifications affecting enzyme activity

• Host-pathogen interface:

  • Study how host metabolites affect bacterial GMP synthase activity

  • Analyze competition for nucleotide precursors between host and pathogen

  • Identify metabolic bottlenecks during infection that could be therapeutic targets

This integrative perspective would place GMP synthase research in a broader biological context, potentially revealing unexpected connections and novel intervention strategies for controlling E. ictaluri infections in aquaculture.