Recombinant Burkholderia multivorans GMP synthase [glutamine-hydrolyzing] (guaA), partial

Basic Research Questions

• What is GMP synthase (guaA) and what role does it play in Burkholderia multivorans metabolism?

GMP synthase [glutamine-hydrolyzing] (guaA) catalyzes the final step in de novo GMP biosynthesis, converting xanthosine 5′-monophosphate (XMP) to guanosine 5′-monophosphate (GMP) using glutamine as an amino group donor and ATP as an energy source. This reaction proceeds through an adenyl-XMP intermediate . In B. multivorans, as in other bacteria, GMP synthase plays an essential role in nucleotide metabolism, providing GMP for DNA replication, transcription, and translation. GMP serves as the precursor to GTP, which supports numerous cellular processes and functions as an energy source . The enzyme belongs to the glutamine amidotransferase (GAT) family, which characteristically hydrolyzes glutamine and transfers the generated ammonia to various metabolites . GMP synthase's essentiality in many pathogenic bacteria makes it a potential antimicrobial target, similar to findings in Mycobacterium tuberculosis where the enzyme has been shown to be required for in vitro growth .

• What is the structure and catalytic mechanism of GMP synthase?

GMP synthase exists as a homodimer with each monomer consisting of three distinct domains: a class I glutamine amidotransferase (GATase) domain, an ATP pyrophosphatase (ATPPase) domain, and a dimerization domain . The catalytic mechanism involves two separate but coordinated reactions:

• Glutaminase activity: The GATase domain hydrolyzes glutamine to glutamate and ammonia using a conserved catalytic triad (Cys-His-Glu) . The cysteine residue (equivalent to Cys104 in human GMP synthetase) acts as the nucleophile essential for glutamine hydrolysis .

• Synthetase activity: The ATPPase domain catalyzes ATP-dependent adenylation of XMP to form an adenyl-XMP intermediate, followed by amination to produce GMP .

These two reactions are coordinated through an ammonia tunnel connecting the two active sites. This structural feature ensures that ammonia generated from glutamine hydrolysis is channeled directly to the ATPPase site without equilibrating with the external medium . The binding of substrates (ATP·Mg²⁺ and XMP) to the ATPPase domain allosterically activates the glutaminase activity, ensuring proper synchronization of the two reactions .

• What are the optimal storage and handling conditions for recombinant B. multivorans GMP synthase?

For optimal preservation of enzymatic activity, recombinant B. multivorans GMP synthase should be stored at -20°C for routine use. For extended storage, maintain the protein at -20°C or -80°C . Repeated freezing and thawing should be avoided as this can significantly compromise enzyme activity. Working aliquots may be stored at 4°C for up to one week .

For reconstitution of lyophilized protein:

• Briefly centrifuge the vial before opening to collect contents at the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

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

The shelf life varies based on storage conditions: approximately 6 months for liquid formulations at -20°C/-80°C, and 12 months for lyophilized preparations at the same temperatures .

• What kinetic parameters characterize GMP synthase activity across different species?

Although specific kinetic parameters for B. multivorans GMP synthase are not directly available from the search results, comparative data from other organisms provides valuable context:

Notably, substrate kinetics vary significantly between species, with XMP exhibiting sigmoidal kinetics (indicating cooperativity) in human and M. tuberculosis enzymes, while showing hyperbolic kinetics in other organisms . These differences might be exploited for species-specific targeting in drug development.

• How is GMP synthase activity regulated in bacterial systems?

GMP synthase activity is regulated through multiple mechanisms to maintain appropriate guanine nucleotide levels. In bacterial systems like Clostridioides difficile, guaA expression is controlled by a guanine-responsive riboswitch that senses intracellular guanine levels . This regulatory mechanism enables bacteria to adjust GMP synthesis based on cellular needs.

At the enzyme level, regulation involves substrate-induced activation, where binding of ATP·Mg²⁺ and XMP to the ATPPase domain allosterically activates the glutaminase activity . This coordination ensures that glutamine hydrolysis occurs only when the synthetase domain is ready to utilize the ammonia generated. Inorganic pyrophosphate acts as an inhibitory regulator, uncoupling the two domain functions by allowing glutamine hydrolysis to occur without ATP hydrolysis or GMP formation . This feedback mechanism helps maintain balanced nucleotide metabolism by preventing wasteful consumption of glutamine when GMP production is inhibited.

Advanced Research Questions

• What methodologies are most effective for assessing inhibitor efficacy against B. multivorans GMP synthase?

A comprehensive approach to evaluating inhibitor efficacy against B. multivorans GMP synthase should incorporate multiple complementary methodologies:

• Enzymatic assays:

  • Measure glutaminase activity by quantifying glutamate production using glutamate dehydrogenase coupling

  • Assess ATPPase activity by monitoring ATP hydrolysis through coupled enzyme systems (pyruvate kinase/lactate dehydrogenase)

  • Determine GMP formation directly using HPLC or spectrophotometric methods

• Inhibition characterization:

  • Determine IC50 values using dose-response curves

  • Perform detailed kinetic analysis to classify inhibition mechanisms (competitive, non-competitive, uncompetitive, or mixed)

  • Evaluate time-dependent inhibition to identify potentially irreversible inhibitors similar to acivicin

• Binding studies:

  • Employ isothermal titration calorimetry (ITC) to determine binding thermodynamics

  • Use surface plasmon resonance (SPR) to measure binding kinetics

  • Perform thermal shift assays to assess inhibitor-induced stability changes

• Selectivity assessment:

  • Compare inhibition profiles against human GMP synthase to ensure selectivity

  • Test against related enzymes to determine specificity within the glutamine amidotransferase family

• Structural validation:

  • Conduct co-crystallization or soaking experiments to determine inhibitor binding modes

  • Perform molecular dynamics simulations to understand inhibitor interactions

When analyzing glutamine analogs like acivicin, it's important to test inhibition using both glutamine and ammonia as amino donors. An effective glutaminase-specific inhibitor will block activity only when glutamine is the donor, while ammonia-dependent activity should remain unaffected . This pattern was observed with acivicin, which covalently modifies the catalytic cysteine residue in the glutaminase domain .

• How can site-directed mutagenesis be designed to probe the ammonia channeling mechanism in B. multivorans GMP synthase?

Site-directed mutagenesis represents a powerful approach to investigate the ammonia channeling mechanism that connects the glutaminase and synthetase activities in GMP synthase. A systematic experimental design should target residues hypothesized to participate in different aspects of channel function:

The functional impact of these mutations should be assessed through:

• Enzymatic activity measurements:

  • Compare glutaminase activity alone versus coupled GMP formation

  • Calculate coupling efficiency (ratio of glutamine hydrolyzed to GMP formed)

  • Measure ammonia-dependent versus glutamine-dependent GMP synthesis rates

• Structural analysis:

  • Determine crystal structures of key mutants to visualize channel alterations

  • Perform molecular dynamics simulations to model ammonia movement through altered channels

A successful experiment would identify mutants with decoupled activities, where glutamine hydrolysis occurs normally but GMP formation is impaired, or mutations that alter the kinetics of coupled reactions. The comparison between glutamine-dependent and ammonia-dependent activities provides a critical control, as mutations affecting only the glutamine-dependent reaction likely disrupt the channeling mechanism .

• What approaches can be used to investigate the allosteric communication between domains in B. multivorans GMP synthase?

Investigating the allosteric communication between the glutaminase and synthetase domains of B. multivorans GMP synthase requires a multifaceted approach combining structural, biochemical, and biophysical techniques:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium incorporation patterns in the presence and absence of substrates

  • Identify regions with altered solvent accessibility upon substrate binding

  • Map dynamic changes that propagate from one domain to another

• Truncation and chimeric protein studies:

  • Create isolated domain constructs to assess intrinsic activities

  • Design chimeric proteins by swapping domains between species to identify species-specific allosteric mechanisms

  • Analyze the reconstitution of activity when separated domains are mixed

• Cross-linking studies:

  • Apply zero-length or short cross-linkers in different substrate-bound states

  • Identify residue pairs that come into proximity during conformational changes

  • Use mass spectrometry to map cross-linked residues

• Fluorescence-based approaches:

  • Introduce site-specific fluorophores at domain interfaces

  • Monitor changes in fluorescence intensity or FRET efficiency upon substrate binding

  • Track real-time conformational changes during catalysis

• Computational analysis:

  • Perform normal mode analysis to identify correlated motions between domains

  • Use molecular dynamics simulations to model allosteric signal propagation

  • Apply network analysis to identify residue communication pathways

A particularly insightful experiment would involve creating a series of point mutations at the domain interface and measuring the effect on substrate-induced activation of the glutaminase domain. The search results indicate that binding of ATP·Mg²⁺ and XMP to the ATPPase domain allosterically activates the glutaminase domain , suggesting the existence of a communication pathway between the domains. Mutations disrupting this pathway would be expected to uncouple the two activities, allowing independent glutaminase activity or altering the substrate concentration dependence of activation.

• How can isotope labeling studies be designed to track reaction intermediates in the B. multivorans GMP synthase mechanism?

Isotope labeling studies provide powerful tools for elucidating enzyme reaction mechanisms by tracking atoms through catalytic pathways. For B. multivorans GMP synthase, several strategic approaches can reveal key mechanistic details:

• Ammonia transfer tracking with ¹⁵N-labeled glutamine:

  • React enzyme with [¹⁵N]glutamine, ATP, XMP, and Mg²⁺

  • Isolate GMP product and analyze by NMR or mass spectrometry

  • The appearance of ¹⁵N in GMP confirms direct transfer of the glutamine amide nitrogen

  • Absence of measurable ¹⁵N-ammonia in the reaction medium would support the channeling hypothesis

• Adenyl-XMP intermediate detection using positional isotope exchange:

  • Employ [γ-¹⁸O]ATP as substrate

  • Monitor ¹⁸O distribution in reaction products and remaining ATP by ³¹P NMR

  • Formation of the adenyl-XMP intermediate would result in specific patterns of ¹⁸O scrambling

• Reaction coordinate analysis using kinetic isotope effects:

  • Compare reaction rates with [¹⁵N]glutamine versus unlabeled glutamine

  • Measure kinetics with ¹⁸O-labeled XMP at the site of amination

  • Calculate primary and secondary isotope effects to identify rate-limiting steps

• Water participation assessment:

  • Conduct reactions in H₂¹⁸O buffer

  • Analyze products for ¹⁸O incorporation

  • Determine if water molecules participate in hydrolysis or nucleophilic steps

• Trapping reaction intermediates:

  • Use substrate analogs that allow formation but prevent turnover of intermediates

  • Rapidly quench reactions at different time points

  • Analyze trapped species by mass spectrometry or NMR

A particularly informative experiment would combine isotope labeling with time-resolved techniques to capture the sequence and timing of chemical transformations. For example, rapid-quench flow methods coupled with mass spectrometry analysis could track the formation and decay of the adenyl-XMP intermediate, while pulse-chase experiments with labeled and unlabeled substrates could reveal the commitment to catalysis at various stages of the reaction.

• What structure-function relationships in GMP synthase might be exploited for selective inhibitor design targeting B. multivorans?

Developing selective inhibitors against B. multivorans GMP synthase requires identifying and targeting structural features that differ from human counterparts. Several promising approaches emerge from analysis of GMP synthase structure-function relationships:

• Targeting the ammonia tunnel:

  • The ammonia channel connecting the glutaminase and synthetase domains represents a unique structural feature

  • Species-specific residues lining this tunnel could be targeted by small molecules that block ammonia transfer

  • Compounds that disrupt the conformational changes required for channel formation would selectively inhibit the coupled reaction

• Exploiting domain interface differences:

  • The allosteric communication between domains likely involves species-specific residue networks

  • Inhibitors that bind at the domain interface could prevent the conformational changes needed for substrate-induced activation

  • Differences in interface architecture between bacterial and human enzymes offer selectivity potential

• Adenyl-XMP intermediate stabilization:

  • Compounds that mimic the adenyl-XMP intermediate but contain non-hydrolyzable bonds could act as transition-state inhibitors

  • Species-specific residues in the active site that interact with this intermediate could be targeted for selective binding

• Glutamine binding pocket exploitation:

  • Despite the conserved catalytic triad, surrounding residues that influence glutamine binding specificity may differ between species

  • Glutamine analogs with selective modifications to interact with bacterial-specific residues could achieve selectivity

• Allosteric site targeting:

  • Identify bacterial-specific allosteric sites that regulate enzyme activity

  • Develop modulators that bind these sites without affecting the human enzyme

The most promising strategy likely involves a structure-based approach that first identifies key differences in binding pockets or dynamic features between B. multivorans and human GMP synthases. Acivicin provides an instructive example as a glutamine analog that irreversibly inhibits glutaminase activity by covalently modifying the catalytic cysteine residue (Cys104 in human enzyme) . Modified versions of such inhibitors could be designed to interact preferentially with the bacterial enzyme's specific binding environment.

• How does substrate binding regulate conformational dynamics in GMP synthase, and how can this be investigated?

Substrate binding induces critical conformational changes in GMP synthase that coordinate its dual catalytic activities. These dynamic structural transitions represent both a fundamental mechanistic question and potential targets for inhibitor development.

The binding of ATP·Mg²⁺ and XMP to the ATPPase domain triggers conformational changes that allosterically activate the glutaminase domain, increasing its affinity for glutamine and enhancing glutaminase activity . This substrate-induced activation ensures that glutamine hydrolysis occurs only when the synthetase domain is prepared to utilize the ammonia produced. Additionally, substrate binding leads to the formation of the ammonia tunnel connecting the two active sites .

To investigate these conformational dynamics, several complementary approaches can be employed:

• Time-resolved structural methods:

  • Time-resolved X-ray crystallography using temperature-jump or substrate-release triggers

  • Time-resolved cryo-electron microscopy to capture transient conformational states

  • Small-angle X-ray scattering (SAXS) to monitor global conformational changes in solution

• Single-molecule techniques:

  • Single-molecule FRET with strategically placed fluorophores to track domain movements

  • Optical tweezers to measure force generation during conformational changes

  • High-speed atomic force microscopy to visualize structural dynamics in real-time

• Spectroscopic methods:

  • Stopped-flow spectroscopy coupled with intrinsic tryptophan fluorescence

  • Circular dichroism spectroscopy to detect secondary structure changes

  • NMR relaxation dispersion experiments to identify millisecond timescale motions

• Computational approaches:

  • Enhanced sampling molecular dynamics to model conformational transitions

  • Markov state modeling to identify key intermediate states

  • Elastic network models to predict dominant modes of collective motion

A particularly revealing experimental design would combine site-specific fluorescent labeling with stopped-flow kinetics to correlate conformational changes with individual catalytic steps. By introducing pairs of fluorophores at domain interfaces and monitoring FRET efficiency changes upon sequential addition of substrates, researchers could determine the order and rate of conformational changes, potentially identifying rate-limiting structural transitions that could be targeted by inhibitors.

• What potential roles does GMP synthase play in Burkholderia multivorans pathogenicity and antibiotic resistance?

While direct evidence for GMP synthase's role in B. multivorans pathogenicity is limited in the search results, informed extrapolation from related bacterial systems suggests several potential connections:

• Contribution to bacterial fitness during infection:

  • As a key enzyme in guanine nucleotide biosynthesis, GMP synthase likely supports the rapid growth required during infection

  • In nutrient-limited host environments where salvage pathways may be insufficient, de novo GMP synthesis becomes critical

  • Similar to findings in M. tuberculosis, GMP synthase may be essential for B. multivorans growth in vivo

• Potential involvement in biofilm formation:

  • Burkholderia species are known for forming robust biofilms that contribute to persistence

  • Nucleotide metabolism enzymes have been implicated in biofilm formation in other bacteria

  • Guanine nucleotides serve as precursors to cyclic-di-GMP, a key bacterial second messenger regulating biofilm formation

• Stress response and adaptation:

  • Proper nucleotide pool maintenance is crucial during stress responses

  • GMP synthase activity may be modulated during host-imposed stresses

  • Adaptive regulation of GMP synthesis could contribute to persistence

• Relationship to antibiotic resistance:

  • Altered nucleotide metabolism has been linked to antibiotic tolerance in some bacteria

  • Changes in GMP synthase expression or activity could potentially influence susceptibility to certain antibiotics

  • The enzyme may indirectly contribute to resistance by supporting growth under antibiotic pressure

• Interactions with host immune system:

  • Bacterial nucleotide metabolism enzymes can influence host-pathogen interactions

  • Metabolites produced or consumed by GMP synthase might modulate host responses

The Burkholderia cepacia complex, which includes B. multivorans, is known to produce various virulence factors including hemolytic peptides termed "cepalysins" and other bioactive compounds . While not directly linked to GMP synthase in the search results, these factors demonstrate the complex pathogenicity mechanisms employed by these bacteria. Further research exploring potential connections between GMP synthase activity and the expression or function of such virulence factors could reveal novel aspects of B. multivorans pathogenesis.