Recombinant Desulfovibrio salexigens GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is GMP synthase in Desulfovibrio salexigens and what role does it play in cellular metabolism?

GMP synthase (EC 6.3.5.2), encoded by the guaA gene in Desulfovibrio salexigens, is a critical enzyme that catalyzes the amination of xanthosine 5'-monophosphate (XMP) to form guanosine monophosphate (GMP) in the presence of glutamine and ATP. This reaction represents the final step in the de novo synthesis of guanine nucleotides.

The reaction can be summarized as:
ATP + XMP + L-glutamine + H₂O → AMP + diphosphate + GMP + L-glutamate

The enzyme contains two functional domains that work in coordination:

• The glutaminase domain (N-terminal): Responsible for glutamine hydrolysis to produce the necessary amino group

• The synthetase domain (C-terminal): Responsible for ATP hydrolysis and the transfer of the amino group to XMP

In D. salexigens, GMP synthase plays a crucial role in purine metabolism, which is essential for genetic material synthesis, energy transfer processes, and various signaling pathways. Unlike in some organisms, D. salexigens must efficiently manage its nucleotide synthesis under anaerobic conditions, making the study of its GMP synthase particularly interesting for understanding metabolic adaptations in sulfate-reducing bacteria .

How does the structure of GMP synthase from D. salexigens differ from other bacterial homologs?

The structural characteristics of D. salexigens GMP synthase show both conserved elements and unique adaptations compared to other bacterial homologs:

Conserved Features:

• Like other GMP synthases, it contains both glutaminase and synthetase domains

• The catalytic triad in the Class I amidotransferase domain (typically Cys, His, Glu) is preserved

• The ATP-binding P-loop motif in the synthetase domain is structurally conserved

Distinctive Features:

• Amino acid composition analysis between D. salexigens and other bacteria reveals statistically significant substitutions in several amino acids, particularly aspartic acid (D), glutamic acid (E), lysine (K), asparagine (N), serine (S) and tyrosine (Y)

• These amino acid preferences likely reflect adaptations to D. salexigens' unique marine and anaerobic lifestyle

These differences likely contribute to optimized enzyme function under the specific physiological conditions of D. salexigens, including potential adaptations for anaerobic environments and marine salinity .

What expression systems are most effective for producing active recombinant D. salexigens GMP synthase?

For successful expression of recombinant D. salexigens GMP synthase, several expression systems have been evaluated, with E. coli-based systems showing particular promise:

E. coli Expression Systems:

• Bactosome expression systems in E. coli have demonstrated excellent results for recombinant enzyme production

• E. coli BL21(DE3) RIL strains are particularly effective as they provide additional tRNAs for rare codons that may be present in D. salexigens genes

Key Methodological Considerations:

• Vector selection: pET-based vectors with T7 promoters provide high-level expression control

• Induction parameters: Optimal results typically achieved with 0.5-1.0 mM IPTG induction at OD₆₀₀ of 0.6-0.8

• Growth temperature: Post-induction temperature reduction to 16-20°C often improves soluble protein yield

• Anaerobic considerations: Since D. salexigens is anaerobic, expression under microaerobic conditions may improve proper folding

Purification Approach:

• Metal affinity chromatography using His-tagged constructs yields high purity

• Size exclusion chromatography can further enhance homogeneity

• Enzyme activity is best preserved when purification buffers contain:

  • 10-20% glycerol

  • 1-5 mM DTT or other reducing agents

  • Protease inhibitors during initial extraction

Importantly, when expressing recombinant D. salexigens proteins in E. coli, researchers should verify that no endogenous ligands are co-purified, as mass spectrometry analysis has revealed that E. coli expression can sometimes result in co-purification of native metabolites .

How can enzyme kinetics of D. salexigens GMP synthase be accurately measured under anaerobic conditions?

Measuring enzyme kinetics of D. salexigens GMP synthase under anaerobic conditions requires specialized techniques due to the oxygen sensitivity of this sulfate-reducing bacterium:

Experimental Setup:

• Anaerobic chamber preparation: Use an anaerobic chamber with N₂/H₂/CO₂ atmosphere (typically 85:10:5)

• Buffer degassing: All reaction buffers must be thoroughly degassed and equilibrated in the anaerobic chamber for at least 24 hours

• Enzyme stabilization: Include reducing agents like DTT (1-5 mM) or sodium dithionite to maintain reducing conditions

Kinetic Measurement Methods:

• Coupled enzyme assays:

  • Monitor AMP production by coupling with adenylate kinase and pyruvate kinase/lactate dehydrogenase

  • Follow NADH oxidation spectrophotometrically at 340 nm

• Direct product quantification:

  • HPLC analysis of GMP production using anion exchange chromatography

  • Mass spectrometry for precise quantification of reaction products

Important Considerations:

• Control experiments must verify that coupled enzymes remain active under anaerobic conditions

• Desulfovibrio enzymes may show inactivation during oxygen exposure, similar to NADH oxidase inactivation observed in D. salexigens Mast1

• Enzyme activity should be measured immediately after purification as storage can lead to activity loss

Data Analysis Table Example:

Note: Values are representative ranges based on typical bacterial GMP synthases and should be experimentally determined for D. salexigens specifically

What is the role of conserved cysteine residues in the catalytic mechanism of D. salexigens GMP synthase?

Conserved cysteine residues play crucial roles in the catalytic mechanism of D. salexigens GMP synthase, particularly in the glutaminase domain:

Catalytic Mechanism:

• The glutaminase domain contains a conserved catalytic triad, with a cysteine residue (equivalent to Cys104 in human GMP synthase) as the key nucleophile

• This cysteine initiates glutamine hydrolysis by nucleophilic attack on the amide carbon of glutamine

• The resulting intermediate releases the amino group, which is then channeled to the synthetase domain

• The amino group subsequently attacks XMP (activated by ATP) to form GMP

Evidence for Cysteine Importance:

• Studies with recombinant GMP synthase have shown that modification of the equivalent cysteine with acivicin (a glutamine analog) irreversibly inhibits the glutaminase activity

• Mass spectrometry and Edman sequence analysis have confirmed that this cysteine is the site of acivicin modification

• Mutation studies demonstrate that altering this cysteine residue (e.g., Cys to Ala) abolishes glutaminase activity

Domain Coordination:

• The cysteine-dependent glutaminase activity is tightly coordinated with the synthetase domain

• Inorganic pyrophosphate can inhibit the synthetase activity and uncouple the two domain functions, allowing glutamine hydrolysis to proceed without GMP formation

• This suggests that the cysteine-mediated glutamine hydrolysis can operate independently but is normally regulated by the activity of the synthetase domain

This mechanism is likely conserved in D. salexigens GMP synthase, though specific studies on this organism's enzyme would be needed to confirm the exact position and role of the catalytic cysteine.

How does c-di-GMP signaling potentially interact with GMP synthase activity in D. salexigens?

Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger that controls various cellular functions, and there are several potential interactions between c-di-GMP signaling and GMP synthase activity in D. salexigens:

Potential Regulatory Interactions:

• Substrate Competition:

  • GMP is required for c-di-GMP synthesis by diguanylate cyclases (DGCs)

  • GMP synthase activity may therefore directly impact c-di-GMP pool availability

  • Under conditions of high c-di-GMP synthesis, feedback inhibition of GMP synthase could occur

• Transcriptional Regulation:

  • c-di-GMP binding proteins like FleQ function as transcription regulators in many bacteria

  • Similar regulators in D. salexigens could potentially control guaA gene expression

  • Recent discoveries of c-di-GMP receptor proteins such as ComFB (present in many bacteria) suggest additional regulatory possibilities

• Metabolic Coordination:

  • c-di-GMP levels in bacteria are known to respond to environmental conditions such as osmolarity

  • D. salexigens, as a halophilic organism, may coordinate GMP synthase activity with osmotic stress responses

  • This coordination would ensure appropriate nucleotide availability during stress adaptation

Experimental Evidence from Related Systems:

• In other bacteria, c-di-GMP binds to specific motifs in various proteins, affecting their function

• FRET-based c-di-GMP biosensors have demonstrated variable affinity (KD ranging across 100-fold differences) in different bacterial species

• Environmental factors such as NaCl concentration can significantly impact c-di-GMP levels in bacteria, potentially affecting all GMP-dependent pathways

While direct evidence for c-di-GMP regulation of GMP synthase in D. salexigens is currently limited, the interconnection between these pathways presents an interesting area for future investigation, particularly given D. salexigens' requirements for adaptation to osmotic stress.

What methods are most effective for assessing the substrate specificity of D. salexigens GMP synthase?

Assessing substrate specificity of D. salexigens GMP synthase requires a multi-faceted approach combining biochemical, structural, and computational methods:

Biochemical Approaches:

• Alternative Substrate Screening:

  • Test activity with XMP analogs (e.g., 8-azaXMP, 6-thioXMP)

  • Evaluate different amino group donors beyond glutamine (e.g., ammonia, methylamine)

  • Examine ATP analogs (e.g., GTP, CTP) as phosphate donors

• Kinetic Parameter Determination:

  • Measure Km, kcat, and kcat/Km for each potential substrate

  • Compare catalytic efficiency ratios to establish preference hierarchy

  • Evaluate competitive inhibition patterns between substrate analogs

Structural Methods:

• X-ray Crystallography:

  • Obtain crystal structures with various bound substrates or substrate analogs

  • Identify key binding residues and interaction patterns

  • Compare active site architecture with other characterized GMP synthases

• NMR Studies:

  • Characterize protein-substrate interactions in solution

  • Examine dynamic changes upon substrate binding

  • Identify allosteric effects of substrate binding

Computational Approaches:

• Molecular Docking:

  • In silico screening of potential substrates

  • Calculation of binding energies and interaction patterns

  • Prediction of productive binding modes

• Molecular Dynamics Simulations:

  • Model substrate entry and product release pathways

  • Evaluate conformational changes during catalysis

  • Identify water-mediated interactions critical for specificity

Example Data Presentation:

Note: Table contains representative values; actual measurements for D. salexigens GMP synthase would need to be experimentally determined

These complementary approaches provide a comprehensive understanding of substrate specificity, which is particularly important for D. salexigens as it may exhibit adaptations in substrate preference related to its unique ecological niche.

How does the amino acid composition of D. salexigens GMP synthase contribute to its adaptation to marine environments?

The amino acid composition of D. salexigens GMP synthase reflects specific adaptations to marine environments, particularly related to salt tolerance and pressure adaptation:

Halophilic Adaptations:

• Acidic Residue Enrichment:

  • D. salexigens shows statistically significant substitution patterns favoring acidic amino acids (glutamic acid, aspartic acid)

  • These negatively charged residues create a hydration shell that maintains protein solubility in high-salt conditions

  • The exact substitution percentages (glutamic acid: 6.82%, aspartic acid: 2.33%) suggest moderate halophilic adaptation

• Lysine Preference:

  • High lysine substitution rate (13.10%) compared to related non-marine species

  • Positively charged lysines can interact with surrounding water, enhancing hydration

  • May counterbalance negative charges to maintain optimal electrostatic properties

Structural Stability Features:

Comparative Analysis:
Phylogenetic studies place D. salexigens in an isolated branch within the Desulfovibrio genus, suggesting unique evolutionary adaptations . When comparing amino acid compositions between D. salexigens and non-marine Desulfovibrio species, clear patterns emerge:

These compositional differences likely extend to GMP synthase, allowing the enzyme to function efficiently in the marine environment where D. salexigens naturally occurs. This adaptation is crucial for maintaining essential nucleotide synthesis pathways under conditions that might otherwise compromise enzyme structure and function.

How does recombinant expression affect the post-translational modifications of D. salexigens GMP synthase?

Recombinant expression of D. salexigens GMP synthase can significantly impact its post-translational modifications (PTMs), affecting both structural integrity and enzymatic function:

PTM Considerations in Native vs. Recombinant Systems:

• Phosphorylation Patterns:

  • ABC transporters and related enzymes in bacteria can be modified by phosphorylation

  • Recombinant expression in E. coli may result in different kinase activity compared to native D. salexigens

  • Phosphorylation differences could alter enzyme regulation and protein-protein interactions

• Disulfide Bond Formation:

  • The reducing environment in D. salexigens (anaerobic sulfate-reducer) differs from expression hosts

  • Critical cysteine residues in the glutaminase domain may form inappropriate disulfide bonds in aerobic expression systems

  • This could significantly impact the nucleophilic activity of catalytic cysteines

• Proteolytic Processing:

  • N-terminal or C-terminal processing that occurs in D. salexigens may be absent in recombinant systems

  • Fusion tags (His, GST, etc.) may interfere with native folding or processing

  • Terminal extensions could disrupt domain interactions critical for coordinated enzyme function

Experimental Approaches to Address PTM Differences:

• Mass Spectrometry Analysis:

  • Compare PTM profiles between native and recombinant enzymes

  • Identify specific modification sites that differ between systems

  • Quantify modification stoichiometry at each site

• Activity Restoration Strategies:

  • In vitro modification to mimic native PTMs (e.g., treatment with specific kinases)

  • Co-expression with D. salexigens modification enzymes

  • Expression under conditions that promote native-like modifications

• Expression System Optimization:

  • Use of anaerobic expression conditions

  • Expression in related Desulfovibrio species rather than E. coli

  • Development of cell-free expression systems with controlled redox environments

Methodological Table for PTM Analysis:

Understanding and addressing these PTM differences is critical for ensuring that recombinant D. salexigens GMP synthase accurately represents the native enzyme's properties and activities in experimental studies.

What comparative genomic insights can be gained from studying guaA genes across the Desulfovibrionaceae family?

Comparative genomic analysis of guaA genes across the Desulfovibrionaceae family provides valuable insights into evolutionary relationships, functional conservation, and adaptative strategies:

Phylogenetic Relationships:

• The Desulfovibrionaceae family contains several distinct lineages with varying degrees of relatedness

• D. salexigens forms an isolated branch in phylogenetic analyses, suggesting a unique evolutionary history

• guaA gene sequences can be used as molecular markers to clarify taxonomic relationships within this complex bacterial family

Gene Structure and Organization:

• Analysis of guaA and surrounding genomic regions reveals conservation patterns:

  • Variations in operon structure across species

  • Presence/absence of regulatory elements

  • Co-occurrence with specific metabolic genes

• D. gigas, a related species, has a remarkably compact genome with a gene density of 1128 bp per gene and average gene length of 993 bp , providing context for analyzing guaA organization in D. salexigens

Functional Adaptations:

Horizontal Gene Transfer Analysis:

• Examination of guaA codon usage and GC content can reveal potential horizontal gene transfer events

• The presence of unique insertion sequences or mobile genetic elements near guaA in some species suggests evolutionary dynamics

• Comparison with plasmid sequences (like D. gigas' 101,949 bp plasmid ) can identify potential mobilization of metabolic genes

Adaptive Variation:

• Amino acid substitution patterns in GMP synthase reflect environmental adaptations:

  • Halophilic adaptations in marine species

  • Temperature adaptations in thermophilic members

  • Pressure adaptations in deep-sea isolates

• These patterns provide insight into the evolutionary pressures shaping nucleotide metabolism in different ecological niches

This comparative analysis not only clarifies the evolutionary history of D. salexigens and related species but also provides insights into the functional adaptation of fundamental metabolic enzymes across diverse environments.

What methodological approaches are most effective for studying the interaction between D. salexigens GMP synthase and potential inhibitors?

Investigating interactions between D. salexigens GMP synthase and potential inhibitors requires a multi-faceted approach combining biophysical, biochemical, and computational methods:

High-Throughput Screening Approaches:

• Targeted Screening of Compound Libraries:

  • Utilize differential scanning fluorimetry (DSF) to monitor thermal shifts upon inhibitor binding

  • Develop activity-based assays suitable for 96/384-well format

  • Screen focused libraries based on known inhibitors of GMP synthases

• Fragment-Based Screening:

  • Use NMR-based methods to identify small molecule fragments that bind

  • Employ surface plasmon resonance (SPR) to quantify binding kinetics

  • Combine fragments to develop higher-affinity inhibitors

Detailed Interaction Analysis:

• Structural Characterization:

  • X-ray crystallography of enzyme-inhibitor complexes

  • Cryo-EM for larger complexes or dynamic states

  • Hydrogen-deuterium exchange mass spectrometry to identify binding-induced conformational changes

• Binding Affinity Determination:

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis for solution-based affinity measurements

  • Fluorescence-based binding assays for high-sensitivity detection

Functional Consequence Assessment:

• Inhibition Mechanism Studies:

  • Determine inhibition type (competitive, non-competitive, uncompetitive, mixed)

  • Measure IC₅₀ and K_i values under various substrate concentrations

  • Evaluate time-dependent inhibition and reversibility

• Domain-Specific Inhibition Analysis:

  • Test inhibitors specific to glutaminase domain (like acivicin)

  • Evaluate ATP-competitive inhibitors for synthetase domain

  • Investigate allosteric inhibitors that disrupt domain communication

Example Inhibition Data Format:

Note: Values are representative based on typical GMP synthase inhibitors; actual measurements for D. salexigens GMP synthase would need to be experimentally determined

Special Considerations for D. salexigens:

• Account for potential redox sensitivity due to D. salexigens' anaerobic nature

• Consider salt tolerance when designing buffer systems for inhibitor testing

• Evaluate inhibitor effectiveness under varying ionic strength conditions to reflect the organism's native environment

This methodological framework enables comprehensive characterization of inhibitor interactions and provides a foundation for potential applications in antimicrobial development or basic research tools.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of D. salexigens GMP synthase?

Site-directed mutagenesis represents a powerful approach for elucidating the catalytic mechanism of D. salexigens GMP synthase by systematically altering specific amino acid residues:

Strategic Target Selection:

• Catalytic Triad Residues:

  • Cysteine residue equivalent to Cys104 in human GMP synthase

  • Conserved histidine that acts as a general base

  • Glutamate residue that stabilizes the histidine

• Substrate Binding Residues:

  • ATP-binding P-loop motif residues

  • XMP coordination residues

  • Glutamine recognition pocket amino acids

• Domain Interface Residues:

  • Amino acids at the glutaminase-synthetase domain junction

  • Residues involved in ammonia channeling

  • Interface stabilization amino acids

Mutagenesis Strategy and Workflow:

• Alanine Scanning:

  • Initial replacement of targeted residues with alanine

  • Identifies essential vs. non-essential residues

  • Provides baseline for further targeted mutations

• Conservative Substitutions:

  • Replace with chemically similar amino acids to probe specific properties

  • Cys→Ser to test importance of sulfhydryl vs. hydroxyl

  • Asp→Glu to evaluate precise spatial requirements

• Radical Substitutions:

  • Charge reversal mutations (Asp→Arg) to test electrostatic requirements

  • Hydrophobic to polar substitutions to probe solvent interactions

  • Proline introductions to test conformational flexibility requirements

Functional Analysis of Mutants:

• Kinetic Parameter Determination:

  • Measure Km, kcat, and kcat/Km for each substrate with each mutant

  • Evaluate changes in catalytic efficiency

  • Determine substrate specificity alterations

• Domain Communication Assessment:

  • Test for uncoupling of glutaminase and synthetase activities

  • Measure glutamine hydrolysis in absence of XMP/ATP

  • Evaluate ammonia channeling efficiency

Example Mutagenesis Results Table:

Note: Table contains representative values based on similar studies; actual measurements for D. salexigens GMP synthase would need to be experimentally determined

Advanced Applications:

• Double and Triple Mutants:

  • Evaluate synergistic effects

  • Test compensatory mutations

  • Reconstruct evolutionary trajectories

• Insertion of Chemical Crosslinkers:

  • Introduce unnatural amino acids with photoactivatable groups

  • Map spatial relationships during catalysis

  • Capture transient interaction states

• pH-Dependent Activity Profiles:

  • Compare pH optima of wild-type and mutants

  • Identify residues involved in proton transfers

  • Determine ionization states critical for catalysis

This systematic mutagenesis approach, combined with thorough functional characterization, can provide detailed insights into the molecular mechanism of D. salexigens GMP synthase and reveal potential adaptations specific to this organism's unique ecological niche.

How does oxygen exposure affect the stability and activity of recombinant D. salexigens GMP synthase?

As D. salexigens is an anaerobic sulfate-reducing bacterium, oxygen exposure has significant impacts on the stability and activity of its recombinant GMP synthase:

Oxygen Sensitivity Mechanisms:

• Cysteine Oxidation:

  • The catalytic cysteine in the glutaminase domain is highly susceptible to oxidation

  • Formation of sulfenic, sulfinic, or sulfonic acid derivatives inactivates the enzyme

  • Potential disulfide bond formation between non-catalytic cysteines disrupts structure

• Metal Center Destabilization:

  • If present, metal cofactors may undergo redox changes upon oxygen exposure

  • Oxidation of iron or other transition metals could affect structural integrity

  • Metal loss following oxidation may irreversibly inactivate the enzyme

• Structural Alterations:

  • Oxidative modifications to amino acid side chains beyond cysteines

  • Potential formation of carbonyl derivatives on sensitive residues

  • Conformational changes that disrupt domain coordination

Experimental Evidence from Related Systems:
Studies with D. salexigens Mast1 demonstrated that oxidation rates were highest at air saturation (up to 40 nmol of O₂ min⁻¹ mg of protein⁻¹) and declined with decreasing oxygen concentrations. Notably, oxidation activity in D. salexigens was found to be entirely dependent on NADH oxidase, which was prone to inactivation as soon as it catalyzed NADH oxidation .

Protective Strategies for Handling Recombinant Enzyme:

• Buffer Optimization:

  • Include reducing agents (5-10 mM DTT, 2-5 mM β-mercaptoethanol)

  • Add oxygen scavengers (glucose oxidase/catalase system)

  • Include metal chelators to prevent Fenton chemistry (EDTA at 1-2 mM)

• Anaerobic Handling Techniques:

  • Purification under strict anaerobic conditions

  • Use of anaerobic chambers for enzyme manipulation

  • Degassing all buffers with inert gas (N₂ or Ar)

• Stabilizing Additives:

  • Glycerol (20-30%) to enhance conformational stability

  • Osmolytes that preferentially hydrate the protein surface

  • Specific substrates or substrate analogs that protect active sites

Activity Recovery Approaches:

Note: Values are representative based on typical oxygen-sensitive enzymes; actual measurements for D. salexigens GMP synthase would need to be experimentally determined

The unique sensitivity of D. salexigens enzymes to oxygen highlights the importance of proper handling techniques when working with recombinant GMP synthase from this organism. These considerations are essential for obtaining reliable activity measurements and structural data.

What are the most promising applications of recombinant D. salexigens GMP synthase in synthetic biology?

Recombinant D. salexigens GMP synthase offers several promising applications in synthetic biology, leveraging its unique properties as an enzyme from a specialized anaerobic marine bacterium:

Engineered Pathway Applications:

• Nucleotide Production Systems:

  • Integration into cell-free systems for sustainable GMP/GTP production

  • Creation of artificial metabolic modules for purine salvage

  • Development of biosensors for nucleotide pool monitoring

• Osmoregulation Engineering:

  • Harnessing D. salexigens' salt tolerance mechanisms

  • Creating systems that link nucleotide metabolism to osmotic stress responses

  • Engineering crops with improved salt tolerance using bacterial osmoregulatory components

• Anaerobic Production Platforms:

  • Designing oxygen-independent nucleotide synthesis pathways

  • Creating metabolic modules functional in anaerobic industrial fermentation

  • Developing bioremediation tools for anaerobic environments

Enzyme Engineering Opportunities:

• Substrate Specificity Modification:

  • Engineering GMP synthase to accept non-canonical substrates

  • Creating variants that produce nucleotide analogs for drug development

  • Developing enzymes with broader or narrower substrate profiles

• Environmental Adaptation Transfer:

  • Identifying and transferring salt-tolerance features to enzymes from non-halophilic organisms

  • Engineering oxygen tolerance while maintaining catalytic efficiency

  • Creating hybrid enzymes with combined beneficial properties

• Protein Architecture Applications:

  • Using the two-domain architecture as a scaffold for enzyme engineering

  • Developing synthetic signaling systems based on domain communication principles

  • Creating novel biocatalysts with coordinated sequential activities

Research Tool Development:

• c-di-GMP Signaling Studies:

  • Tools for manipulating bacterial second messenger pathways

  • Biosensors for monitoring cellular nucleotide dynamics

  • Model systems for studying regulatory networks

• Extremophile Enzyme Models:

  • Platforms for understanding enzyme adaptation to harsh conditions

  • Templates for engineering robust biocatalysts

  • Systems for studying evolutionary trajectories of specialized enzymes

Potential Implementation Approaches: