Recombinant Thermotoga sp. GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the function of GMP synthase (guaA) in Thermotoga species and how does it fit into purine metabolism?

GMP synthase [glutamine-hydrolyzing] (guaA) in Thermotoga species catalyzes a critical step in de novo purine biosynthesis, specifically replacing the exocyclic oxygen at the 2 position of xanthosine monophosphate (XMP) with an amino group to form guanosine monophosphate (GMP) . This enzyme functions as part of an interconnected network of purine metabolism enzymes in Thermotoga.

The complete genome sequencing of Thermotoga species has revealed that their core genome contains several important purine metabolism genes including guaA (GMP synthase), guaB (IMP dehydrogenase), purA (adenylosuccinate synthetase), and purB (adenylosuccinate lyase) . These enzymes work cooperatively to regulate the balance between adenine and guanine nucleotides in the cell.

GMP synthase consists of two functional domains:

• A glutamine amidotransferase domain that hydrolyzes glutamine to produce ammonia

• A synthase domain that transfers the ammonia to XMP to produce GMP

The reaction utilizes ATP as an energy donor, which contributes to balancing the production of guanine nucleotides with adenine nucleotide pools . The guaBA operon in bacteria is organized with guaB preceding guaA, and transcription is regulated by PurR, which represses the operon approximately 5-fold in response to guanine or hypoxanthine .

For experimental investigation of enzyme function, researchers typically employ:

• Spectrophotometric assays monitoring NADH oxidation in coupled enzyme systems

• HPLC analysis of nucleotide production

• Radioactive labeling with 14C-glutamine or 32P-ATP to track reaction progress

What are the optimal conditions for expressing and purifying recombinant Thermotoga sp. guaA?

Successfully expressing and purifying recombinant Thermotoga sp. guaA requires optimization of multiple parameters based on the hyperthermophilic nature of the source organism. The following methodological approach has proven effective:

Expression system:

• Host: Escherichia coli is the preferred heterologous host for expressing Thermotoga proteins

• Vector: Expression vectors containing T7 promoters and appropriate affinity tags (His6 or MBP) enhance purification efficiency

• Strain selection: BL21(DE3) derivatives with rare codon supplementation can improve expression of AT-rich Thermotoga genes

Optimized expression protocol:

• Culture growth: LB medium supplemented with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8

• Induction: IPTG concentration between 0.1-0.5 mM, with temperature shift to 16-30°C

• Post-induction growth: 4-16 hours depending on temperature (longer at lower temperatures)

• Cell harvesting: Centrifugation at 5,000 g for 15 minutes at 4°C

Purification strategy:

• Cell lysis: Sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol

• Heat treatment: Incubation at 70°C for 20 minutes to denature host proteins while retaining thermostable target protein

• Clarification: Centrifugation at 15,000 g for 30 minutes to remove precipitated proteins

• Affinity chromatography: Using appropriate resin based on affinity tag

• Size exclusion chromatography: For higher purity preparations

Storage conditions (based on product data):

• Short-term: 4°C for up to one week

• Long-term: -20°C or -80°C with 5-50% glycerol as cryoprotectant

• Avoid repeated freeze-thaw cycles

Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL with glycerol added to 5-50% final concentration . The shelf life of the liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form can remain stable for 12 months .

How does the thermostability of Thermotoga sp. GMP synthase compare to mesophilic homologs?

Thermotoga species are hyperthermophilic bacteria with optimal growth temperatures of 70-80°C , and their enzymes, including GMP synthase, demonstrate exceptional thermostability compared to mesophilic counterparts. This thermostability represents a crucial adaptation to extreme environments and provides valuable insights for protein engineering.

Comparative thermostability metrics:

Similar to other Thermotoga enzymes, GMP synthase likely achieves thermostability through several structural mechanisms. Studies on Thermotoga maritima indoleglycerol phosphate synthase (tIGPS) have demonstrated that specific solvent-exposed salt bridges significantly contribute to thermostability . In particular, salt bridges that crosslink secondary structure elements separated in sequence but adjacent in the three-dimensional structure provide greater stabilization than those merely tethering terminal regions to the protein core .

Experimental approaches to assess thermostability:

• Thermal inactivation kinetics: Measure residual enzyme activity after incubation at various temperatures (60-100°C) for defined time periods

• Differential scanning calorimetry (DSC): Determine the melting temperature (Tm)

• Circular dichroism (CD) spectroscopy: Monitor secondary structure changes at increasing temperatures

• Intrinsic fluorescence spectroscopy: Track tertiary structure unfolding

• Limited proteolysis: Compare resistance to proteolytic digestion at elevated temperatures

Understanding the molecular basis of thermostability in Thermotoga sp. GMP synthase can inform rational design strategies for engineering mesophilic enzymes with enhanced thermal resistance for biotechnological applications.

What sequence motifs and structural domains are important for the catalytic activity of Thermotoga sp. GMP synthase?

Thermotoga sp. GMP synthase contains several conserved sequence motifs and structural domains critical for its glutamine amidotransferase and synthase activities. Based on the amino acid sequence provided in the product data sheet and comparative analysis with homologous enzymes, the following key features can be identified:

Domain organization:

• N-terminal glutamine amidotransferase (GATase) domain: Responsible for glutamine binding and hydrolysis

• C-terminal synthase domain: Responsible for XMP binding, ATP binding, and amination reaction

Key sequence motifs:

Structural features:

• The glutamine amidotransferase domain contains a catalytic triad (Cys-His-Glu) for glutamine hydrolysis

• The synthase domain adopts an α/β fold with a central β-sheet structure

• An ammonia channel connects the two active sites, allowing transfer of NH3 from the GATase domain to the synthase domain

• ATP binding requires coordination of a Mg²⁺ ion

Experimental approaches to investigate structure-function relationships:

• Site-directed mutagenesis of conserved residues followed by kinetic analysis

• Domain swapping experiments with mesophilic homologs

• X-ray crystallography or cryo-EM to determine the three-dimensional structure

• Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• Molecular dynamics simulations to study substrate binding and catalytic mechanisms

The comparison between Thermotoga sp. GMP synthase and E. coli GMP synthase reveals conservation of catalytic residues while showing differences in surface-exposed regions that likely contribute to thermostability.

How can kinetic modeling be applied to understand the substrate binding and catalytic mechanism of recombinant Thermotoga sp. GMP synthase?

Kinetic modeling provides powerful insights into enzyme mechanisms, substrate binding order, and catalytic efficiency. For Thermotoga sp. GMP synthase, which catalyzes a complex reaction involving three substrates (glutamine, XMP, and ATP), sophisticated kinetic modeling approaches can reveal mechanistic details that are challenging to determine through other methods.

Comprehensive kinetic modeling approach:

• Initial experimental data collection:

  • Measure initial velocities under varying concentrations of all substrates

  • Perform product inhibition studies

  • Determine effects of pH and temperature on reaction rates

• Model development:

  • Propose potential kinetic mechanisms (random, ordered, or ping-pong)

  • Derive rate equations for each mechanism

  • Include parameters for substrate binding (Km values) and catalytic steps (kcat values)

• Parameter estimation:

  • Fit experimental data to candidate models using non-linear regression

  • Use computational tools such as Python-based packages for numerical solutions

  • Determine the most likely mechanism based on statistical fit

Example kinetic model for a three-substrate enzyme:

The full rate equation for a random ter-ter mechanism would be:

$v = \frac{V_{max}[A][B][C]}{K_{ia}K_{b}K_{c} + K_{b}K_{c}[A] + K_{a}K_{c}[B] + K_{a}K_{b}[C] + K_{c}[A][B] + K_{b}[A][C] + K_{a}[B][C] + [A][B][C]}$

Where:

• A, B, and C represent glutamine, XMP, and ATP

• Ka, Kb, and Kc are the respective Michaelis constants

• Kia is the dissociation constant for substrate A

From studies of similar enzymes like GacA , we know that cooperative binding effects can play important roles. The kinetic modeling for GacA revealed KG|A = 10 μM and KA|G = 71 μM, indicating positive cooperativity in substrate binding. Similar analyses for GMP synthase could reveal:

• Order of substrate binding (glutamine → XMP → ATP or alternative sequences)

• Potential cooperative effects between substrates

• Rate-limiting steps in the catalytic cycle

• Effects of physiological substrate concentrations on reaction flux

Using mathematical modeling, researchers can simulate in vivo behavior under cellular conditions, providing insights that may not be apparent from in vitro experiments alone .

What techniques can be used to analyze cooperative binding effects in Thermotoga sp. GMP synthase?

Cooperative binding effects significantly impact enzyme function and can be particularly important for multi-substrate enzymes like GMP synthase. Detecting and characterizing these effects requires specialized experimental techniques and analysis methods.

Comprehensive methodological approach:

• Steady-state kinetic analysis:

  • Generate substrate saturation curves by varying one substrate while keeping others fixed

  • Apply advanced plotting methods to detect non-Michaelis-Menten behavior:

    • Hill plots: log[v/(Vmax-v)] vs. log[S], slope gives Hill coefficient (n)

    • Eadie-Hofstee plots (v vs. v/[S]): curvature indicates cooperativity

  • Determine apparent Km values at different concentrations of the second substrate

• Pre-steady-state kinetics:

  • Use stopped-flow techniques to observe rapid reaction phases

  • Identify binding and conformational change events preceding catalysis

  • Measure rate constants for individual steps in the reaction mechanism

• Biophysical binding assays:

  • Isothermal Titration Calorimetry (ITC): Directly measure binding energetics and stoichiometry

  • Surface Plasmon Resonance (SPR): Monitor real-time binding kinetics

  • Microscale Thermophoresis (MST): Detect binding-induced changes in thermal migration

• Structural and spectroscopic methods:

  • X-ray crystallography with different substrate combinations to visualize binding site changes

  • Nuclear Magnetic Resonance (NMR): Detect conformational changes upon substrate binding

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map regions undergoing conformational changes

• Computational modeling:

  • Similar to the approach used for GacA enzyme , develop mathematical models incorporating:

    • Dissociation constants for each substrate

    • Sequential binding events

    • Conformational changes

  • Simulate product formation under various substrate ratios

  • Compare model predictions with experimental data

For example, research on GacA revealed selective cooperativity where GTP binding was enhanced 2-fold when the enzyme had already bound ATP (KG|A = 10 μM compared to K2G = 20 μM) . Similar analysis of GMP synthase might reveal how binding of glutamine affects subsequent binding of XMP or ATP.

Data analysis approach:

• Plot experimental product ratios against varying substrate concentrations

• Minimize least squares error between model predictions and experimental values to determine optimal binding constants

• Validate model by testing predictions under different experimental conditions

How does the organization and regulation of the guaBA operon in Thermotoga species compare to other bacteria?

Understanding the genomic organization and regulation of the guaBA operon provides insights into the evolutionary adaptations of purine biosynthesis in hyperthermophilic bacteria. While specific information about the guaBA operon in Thermotoga species is limited in the search results, comparative analysis with well-characterized systems can inform research approaches.

Known features of the guaBA operon in E. coli (for comparison):

• Organization: guaB precedes guaA in a bicistronic operon

• Transcription: Occurs from a single promoter

• Regulation: Repressed approximately 5-fold by PurR in response to guanine or hypoxanthine

• Additional regulation: Subject to 15-fold regulation by the DNA initiator protein DnaA

• Structural features: Contains an AT-rich UP-element in the −59 to −38 region that interacts with RNA polymerase α-subunit

Methodological approach for investigating Thermotoga guaBA operon:

• Genomic sequence analysis:

  • Extract and align guaBA regions from multiple Thermotoga genomes (T. maritima, T. petrophila, T. sp. RQ7)

  • Identify gene organization, intergenic regions, and potential regulatory elements

  • Compare with other bacterial phyla to identify conserved and unique features

• Transcriptomic analysis:

  • Perform RNA-Seq under various growth conditions (temperature, carbon source, nutrient limitation)

  • Map transcription start sites and termination sites

  • Determine whether guaB and guaA are co-transcribed

• Promoter characterization:

  • Identify putative promoter elements (-10, -35 boxes, UP elements)

  • Clone promoter regions into reporter constructs

  • Measure activity under varying conditions

• Regulator identification:

  • Search for homologs of known purine regulators (PurR, DnaA) in Thermotoga genomes

  • Perform DNA-protein interaction assays (EMSA, ChIP-seq)

  • Characterize binding sites through footprinting or mutagenesis

Comparative table of guaBA operon features across bacterial species:

Understanding the regulation of guaBA in Thermotoga species would provide insights into how purine biosynthesis is coordinated with other cellular processes in hyperthermophilic environments.

What role does guaA play in the adaptive evolution of Thermotoga species in extreme environments?

Thermotoga species thrive in extreme environments with temperatures ranging from 55-90°C , requiring specialized adaptations at the molecular level. The guaA gene, encoding GMP synthase, provides an excellent model for studying molecular adaptation to extreme conditions, as it performs an essential function common to all life forms while showing specific adaptations in thermophiles.

Methodological approaches to investigate adaptive evolution:

• Comparative sequence analysis:

  • Align guaA sequences from Thermotoga species with mesophilic and other thermophilic homologs

  • Calculate evolutionary rates and selection pressures (dN/dS ratios)

  • Identify sites under positive selection or coevolution networks

  • Construct phylogenetic trees to trace evolutionary history

• Structural bioinformatics:

  • Model 3D structures of GMP synthases from different thermal environments

  • Compare surface charge distribution, hydrogen bonding networks, and hydrophobic cores

  • Identify unique features in Thermotoga GMP synthase that correlate with thermostability

• Experimental evolution:

  • Subject Thermotoga cultures to adaptive laboratory evolution under varying conditions

  • Monitor changes in guaA sequence and expression

  • Characterize functional effects of evolved variants

Key adaptive features likely present in Thermotoga GMP synthase:

The research on indoleglycerol phosphate synthase from T. maritima demonstrated that specific salt bridges, particularly those crosslinking helices separated in sequence but adjacent in structure, contribute significantly to thermostability . Similar principles likely apply to GMP synthase.

The genome sequence of Thermotoga sp. strain RQ7 and comparative analysis with other Thermotoga genomes provides evidence of lateral gene transfer mechanisms that may have contributed to acquisition of thermostable variants or adaptive features . These horizontal gene transfer events could have played a role in the evolution of guaA in Thermotoga species.

How can site-directed mutagenesis be used to investigate the structure-function relationship of Thermotoga sp. GMP synthase?

Site-directed mutagenesis provides a powerful approach to systematically probe the structural features that contribute to catalytic activity, substrate specificity, and thermostability of Thermotoga sp. GMP synthase. A well-designed mutagenesis strategy can reveal key mechanistic insights and guide protein engineering efforts.

Comprehensive mutagenesis approach:

• Target identification:

  • Catalytic residues: Based on sequence alignment with characterized GMP synthases

  • Substrate binding residues: Identified through structural modeling or conserved motifs

  • Thermostability determinants: Focus on surface-exposed salt bridges , buried hydrophobic residues, and loop regions

  • Domain interface residues: Those mediating communication between GATase and synthase domains

• Mutation design strategy:

  • Alanine scanning: Replace targeted residues with alanine to remove side chain function

  • Conservative substitutions: Maintain chemical properties (e.g., Asp→Glu) to test specific requirements

  • Non-conservative substitutions: Change chemical properties to dramatically alter function

  • Thermostability-focused mutations: Replace residues with those found in mesophilic homologs

• Experimental workflow:

  a. Construct generation:

  • PCR-based site-directed mutagenesis (QuikChange or overlap extension PCR)

  • Gibson Assembly for multiple mutations

  • Verification by Sanger sequencing

  b. Protein expression and purification:

  • Express wild-type and mutant proteins under identical conditions

  • Compare expression levels and solubility

  • Apply consistent purification protocol

  c. Functional characterization:

  • Enzyme kinetics: Determine Km, kcat, and kcat/Km for all substrates

  • Thermostability assays: Measure T50, half-life at elevated temperatures

  • Structural analysis: Circular dichroism to assess secondary structure

  • Ligand binding: Isothermal titration calorimetry or fluorescence-based assays

Example mutation targets based on studies of other Thermotoga enzymes:

Studies on indoleglycerol phosphate synthase from T. maritima demonstrated that disrupting salt bridges that crosslink helices α1 and α8 had greater impact on thermostability than disrupting salt bridges tethering the N-terminus . Similar systematic analysis of GMP synthase could identify key stabilizing interactions.

What are the challenges and solutions for maintaining the stability and activity of recombinant Thermotoga sp. GMP synthase during experimental procedures?

Working with enzymes from hyperthermophilic organisms presents unique challenges despite their inherent stability. Recombinant Thermotoga sp. GMP synthase requires specific handling procedures to maintain its structure and function throughout experimental workflows.

Major challenges and methodological solutions:

• Protein storage stability:

  Challenges:

  • Protein aggregation during freeze-thaw cycles

  • Activity loss during extended storage

  Solutions:

  • Store at -20°C or -80°C for long-term preservation

  • Add glycerol (5-50% final concentration) as cryoprotectant

  • Prepare small single-use aliquots to avoid repeated freeze-thaw cycles

  • For working stocks, store at 4°C for up to one week

• Substrate stability at high temperatures:

  Challenges:

  • ATP hydrolysis accelerates at elevated temperatures

  • Glutamine can cyclize or degrade

  • XMP has limited stability in solution

  Solutions:

  • Prepare fresh substrate solutions before each experiment

  • Use higher substrate concentrations to compensate for degradation

  • Account for non-enzymatic substrate degradation with proper controls

  • Consider enzymatic coupled assays that can be performed at lower temperatures

• Buffer considerations:

  Challenges:

  • pH of common buffers changes significantly with temperature

  • Solubility of components may change at high temperatures

  Solutions:

  • Use buffers with minimal ΔpKa/ΔT (e.g., phosphate, HEPES)

  • Adjust pH at the intended working temperature

  • Filter solutions after heating to remove precipitates

  • Include stabilizing additives (e.g., BSA, reducing agents)

• Assay design:

  Challenges:

  • Standard spectrophotometric equipment may not accommodate high temperatures

  • Conventional enzyme assays may not work at optimal temperature for Thermotoga enzymes

  Solutions:

  • Use specialized high-temperature spectrophotometers or sealed cuvettes

  • Develop endpoint assays with heating blocks and rapid cooling

  • Employ activity assays at suboptimal but practical temperatures

  • Extrapolate activity using Arrhenius plots

Practical protocol for reconstitution (based on product data):

• Briefly centrifuge vial before opening to bring contents to bottom

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration (default recommendation: 50%)

• Aliquot for long-term storage at -20°C/-80°C

• For working aliquots, store at 4°C for up to one week

For temperature-activity profiling, measure enzyme activity at 5-10°C intervals from 30-100°C to generate an optimal temperature curve, being mindful that the temperature optimum for activity may differ from the optimal growth temperature of the source organism.