Recombinant Stenotrophomonas maltophilia Glucokinase (glk)

What is the role of glucokinase in S. maltophilia carbohydrate metabolism?

Glucokinase (glk) in S. maltophilia plays a crucial role in carbohydrate metabolism by catalyzing the phosphorylation of glucose to glucose-6-phosphate, the first step in glucose utilization. Unlike many other bacterial species that primarily utilize the phosphotransferase system (PTS), S. maltophilia relies more heavily on glucokinase for glucose metabolism. In S. maltophilia, this enzyme is particularly important because the organism shows a unique ability to metabolize various carbon sources, including galactose and uncommon polyols like l-glucitol . The glk enzyme works in conjunction with other carbohydrate metabolism enzymes, such as d-tagatose 3-epimerase and d-galactose isomerase, to facilitate carbon source utilization in various environmental niches .

How does S. maltophilia glucokinase differ from glucokinases in other bacterial species?

S. maltophilia glucokinase exhibits some distinct characteristics when compared to glucokinases from other bacterial species. While the core catalytic function remains similar, S. maltophilia glk shows adaptation to the organism's metabolic versatility. Analysis of carbohydrate catabolism in S. maltophilia indicates that it possesses unique enzyme sets for glucose metabolism. For instance, S. maltophilia utilizes the Entner-Doudoroff pathway rather than the Embden-Meyerhof-Parnas pathway for glucose metabolism, as evidenced by the absence of d-fructose 6-phosphate kinase and the presence of d-gluconate-6-P dehydratase and 2-keto-3-deoxy-d-gluconate 6-phosphate aldolase . These metabolic differences suggest that S. maltophilia glucokinase may have evolved specific regulatory and catalytic properties to accommodate this alternative glucose metabolism pathway.

What genomic context surrounds the glk gene in S. maltophilia?

The glk gene in S. maltophilia is situated within the context of other carbohydrate metabolism genes. Whole genome sequencing (WGS) and phylogenomic analyses of S. maltophilia strains reveal significant genetic diversity across isolates, with variations in metabolic gene clusters . While specific information about the glk genomic context in the provided search results is limited, comparative genomic analyses of fully finished genomes of S. maltophilia strains (K279a, D457, JV3, R551-3) show that the organization of metabolic genes can vary between strains, potentially affecting glucokinase expression and function . The genetic diversity observed through core SNP phylogenomic analysis suggests that metabolic capabilities, including glucose utilization, may differ among the 12 distinct clades of S. maltophilia identified in recent studies .

What expression systems are most effective for producing recombinant S. maltophilia glucokinase?

For recombinant expression of S. maltophilia glucokinase, E. coli-based expression systems have proven effective. Based on methodologies used for similar S. maltophilia proteins, a typical approach involves cloning the glk gene into expression vectors such as pET series under the control of strong promoters like T7. The methodology adapted from successful expression of other S. maltophilia proteins suggests using BL21(DE3) or similar E. coli strains that provide tight regulation of expression and high protein yields.

The expression protocol should be optimized considering:

• Induction temperature: Often lower temperatures (16-25°C) yield more soluble protein

• Inducer concentration: Typically 0.1-0.5 mM IPTG

• Induction duration: Usually 4-16 hours depending on temperature

When working with challenging expression, alternative approaches might include fusion tags (His6, MBP, GST) to improve solubility and facilitate purification. Codon optimization may be necessary since S. maltophilia has a different codon usage bias compared to E. coli .

What purification strategies yield the highest specific activity for recombinant S. maltophilia glucokinase?

Based on purification approaches for similar metabolic enzymes, a multi-step purification strategy typically yields the highest specific activity for recombinant S. maltophilia glucokinase:

• Initial capture: Affinity chromatography using His-tag (IMAC) if the recombinant glucokinase includes a histidine tag

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose for anion exchange)

• Polishing: Size-exclusion chromatography to obtain homogeneous protein

The purification buffer composition significantly impacts enzyme stability and activity. A typical buffer system might include:

• 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• 100-200 mM NaCl

• 5-10% glycerol (for stability)

• 1-5 mM DTT or β-mercaptoethanol (to maintain reduced state)

• 0.1-1 mM EDTA (to chelate metal ions that might cause oxidation)

Tracking specific activity throughout purification is essential to ensure the protocol maintains enzymatic function. A typical purification table might show:

How can we optimize solubility of recombinant S. maltophilia glucokinase?

Optimizing solubility of recombinant S. maltophilia glucokinase requires addressing several factors:

• Expression temperature: Lower temperatures (16-20°C) slow down protein synthesis, allowing more time for proper folding

• Co-expression with chaperones: GroEL/GroES or DnaK/DnaJ/GrpE chaperone systems can assist in proper folding

• Solubility-enhancing fusion partners: MBP (maltose-binding protein) or SUMO tags significantly enhance solubility

• Buffer composition during lysis and purification:

  • Higher salt concentrations (300-500 mM NaCl)

  • Addition of stabilizing agents like glycerol (10-20%)

  • Non-ionic detergents (0.05-0.1% Triton X-100 or NP-40)

  • Osmolytes such as arginine (50-100 mM) or proline

When dealing with persistent solubility issues, structural analysis of the glucokinase sequence to identify hydrophobic patches that might contribute to aggregation can guide site-directed mutagenesis of non-essential residues to enhance solubility without compromising activity .

What are the key catalytic residues in S. maltophilia glucokinase and how do they compare with other bacterial glucokinases?

The catalytic mechanism of bacterial glucokinases, including S. maltophilia glucokinase, typically involves specific residues in the active site that coordinate with glucose and ATP. While the search results don't provide specific information about S. maltophilia glucokinase catalytic residues, structural homology modeling based on related bacterial glucokinases would suggest conserved residues for substrate binding and catalysis.

Key catalytic features likely include:

• ATP-binding site: Typically contains a conserved motif with lysine and aspartate residues that coordinate with the phosphate groups of ATP

• Glucose-binding site: Often involves polar residues (aspartate, glutamate, asparagine) that form hydrogen bonds with glucose hydroxyl groups

• Catalytic residues: A basic residue (often lysine or arginine) that facilitates the phosphoryl transfer reaction

Comparison with other bacterial glucokinases would likely show conservation of these key functional residues across species, though S. maltophilia might contain unique residues that adapt its activity to the organism's specific metabolic requirements. This adaptation could be particularly relevant given S. maltophilia's distinct carbohydrate metabolism patterns, including the absence of the phosphotransferase system found in many other bacteria .

How does pH and temperature affect the kinetic parameters of S. maltophilia glucokinase?

The activity of S. maltophilia glucokinase, like other enzymes, is significantly influenced by pH and temperature. Optimal conditions reflect the organism's adaptation to its environmental niches.

For temperature effects, a typical kinetic profile might show:

• Activity increases with temperature up to an optimum (likely around 30-37°C, reflecting S. maltophilia's mesophilic nature)

• Activity decreases above the optimum due to thermal denaturation

• Environmental isolates might show lower temperature optima (around 30°C) compared to clinical isolates (37°C)

For pH effects:

• Optimal pH likely falls in the range of 7.0-8.0

• Activity profiles typically follow a bell-shaped curve

• pH affects both substrate binding (Km) and catalytic rate (kcat)

A representative data table for S. maltophilia glucokinase kinetic parameters at different pH values might show:

These parameters are particularly relevant when considering that S. maltophilia can colonize diverse environments with varying pH conditions .

What substrate specificity does S. maltophilia glucokinase exhibit toward different sugars?

S. maltophilia glucokinase likely demonstrates preferential activity toward glucose but may phosphorylate other hexoses at varying rates. Based on the metabolic versatility of S. maltophilia, which can utilize various carbon sources including polyols like L-glucitol , the glucokinase might show broader substrate specificity compared to more specialized bacteria.

A typical substrate specificity profile might show:

This substrate flexibility aligns with the observed metabolic adaptability of S. maltophilia, which enables it to thrive in diverse environmental niches and potentially contributes to its success as an opportunistic pathogen .

How can recombinant S. maltophilia glucokinase be used to study bacterial carbohydrate metabolism pathways?

Recombinant S. maltophilia glucokinase serves as a valuable tool for studying bacterial carbohydrate metabolism pathways, particularly in investigating unique aspects of glucose utilization in this opportunistic pathogen:

• Metabolic flux analysis: Purified recombinant glucokinase can be used in in vitro reconstitution experiments to measure the flux through different carbohydrate metabolism pathways, such as the Entner-Doudoroff pathway that S. maltophilia preferentially uses instead of the Embden-Meyerhof-Parnas pathway .

• Comparative metabolism studies: Since S. maltophilia shows distinct carbohydrate metabolism compared to other bacteria (lacking phosphotransferase systems but having unique enzyme sets), recombinant glucokinase enables direct comparison of the kinetic properties and regulatory mechanisms between different bacterial species.

• Isotope labeling experiments: Using recombinant glucokinase with isotope-labeled glucose substrates allows tracking of carbon flow through metabolic pathways, revealing how S. maltophilia processes different carbon sources.

• Enzyme networks: In vitro studies combining recombinant glucokinase with other enzymes like d-tagatose 3-epimerase and d-galactose isomerase help elucidate how these enzymes work together in carbohydrate metabolism networks, particularly relevant for understanding S. maltophilia's ability to utilize uncommon carbon sources like l-glucitol .

What role might S. maltophilia glucokinase play in biofilm formation and pathogenicity?

While glucokinase's primary role involves metabolism, its function may indirectly impact important S. maltophilia virulence factors like biofilm formation:

• Metabolic adaptation and biofilm formation: Glucokinase activity affects intracellular glucose-6-phosphate levels, which can influence biofilm formation through multiple pathways. S. maltophilia strains show variable biofilm formation capabilities that could correlate with differences in metabolic enzyme expression or activity .

• Energy provision for virulence: Efficient glucose metabolism through glucokinase provides energy necessary for various virulence mechanisms, including the type IV secretion system (T4SS) that S. maltophilia uses for killing competitor bacterial species .

• Adaptation to host environments: Glucokinase activity may help S. maltophilia adapt to glucose-limited environments within human hosts, potentially contributing to its success as an opportunistic pathogen capable of causing healthcare-associated infections .

• Interbacterial competition: By efficiently metabolizing available glucose, S. maltophilia can compete with other microorganisms in polymicrobial infections, complementing its direct bacterial killing mechanisms through the T4SS .

Research suggests that metabolic adaptability is likely a significant factor in S. maltophilia's pathogenicity, as phylogenomic analyses have identified distinct clades of strains that appear specifically adapted to human hosts versus environmental niches .

How can glucokinase activity be measured in complex biological samples from S. maltophilia cultures?

Measuring glucokinase activity in complex biological samples from S. maltophilia cultures requires specific approaches to ensure accuracy and specificity:

• Coupled enzyme assays: The most common approach couples glucokinase activity to glucose-6-phosphate dehydrogenase, which converts G6P to 6-phosphogluconate while reducing NADP+ to NADPH. The NADPH production can be monitored spectrophotometrically at 340 nm, providing a continuous readout of glucokinase activity.

• Radiometric assays: Using [14C]-labeled or [3H]-labeled glucose allows direct measurement of labeled glucose-6-phosphate formation by separation techniques like thin-layer chromatography or ion-exchange chromatography.

• Mass spectrometry-based approaches: LC-MS/MS can be used to directly quantify glucose-6-phosphate production in complex biological samples with high specificity.

• Activity in cell extracts: When measuring glucokinase in S. maltophilia cell extracts, it's important to control for other ATP-consuming processes by using specific inhibitors or differential assays with and without glucose.

A typical protocol for measuring glucokinase activity in S. maltophilia cell extracts might include:

• Buffer: 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2

• Substrates: 10 mM glucose, 5 mM ATP

• Coupling system: 0.5 mM NADP+, 1 U/ml glucose-6-phosphate dehydrogenase

• Controls: Reactions without glucose to account for background ATP consumption

This approach has been successfully used to measure enzyme activities in S. maltophilia extracts, such as d-tagatose 3-epimerase activity by monitoring product formation using HPLC .

How do genetic variations in the glk gene correlate with antimicrobial resistance profiles in S. maltophilia?

The relationship between glk genetic variations and antimicrobial resistance in S. maltophilia represents an emerging area of research:

• Genotype-phenotype correlations: Multilocus variable number of tandem repeat analysis (MLVA) and whole genome sequencing (WGS) studies of S. maltophilia isolates have revealed substantial genetic diversity across strains, with potential implications for metabolic gene function including glk .

• Clade-specific resistance profiles: Phylogenomic analyses have identified distinct S. maltophilia clades, some of which show different antimicrobial resistance profiles. Studies indicate resistance rates to trimethoprim-sulfamethoxazole and levofloxacin can be as high as 30.5%, with ceftazidime resistance at 28.0% .

• Metabolic adaptations and resistance: While direct evidence linking glk variations to antimicrobial resistance is limited, metabolic adaptations often accompany resistance development. Changes in glucose metabolism through altered glucokinase activity could potentially affect:

  • Biofilm formation, which enhances antibiotic tolerance

  • Energy production for efflux pump activity

  • Cell wall synthesis and repair mechanisms

• Clinical vs. environmental isolates: Studies suggest distinct genetic differences between clinical and environmental S. maltophilia isolates, with 6 clades containing exclusively human isolates and 3 clades with exclusively environmental isolates . These genetic differences likely extend to metabolic genes including glucokinase, potentially affecting both virulence and antimicrobial resistance.

What are the potential mechanisms of post-translational regulation of S. maltophilia glucokinase activity?

Glucokinase activity in bacteria, including likely S. maltophilia, is subject to sophisticated post-translational regulation mechanisms:

• Allosteric regulation: Bacterial glucokinases can be regulated by allosteric effectors including metabolic intermediates that signal energy status or carbon availability.

• Phosphorylation: Reversible phosphorylation by protein kinases/phosphatases can modulate glucokinase activity in response to environmental signals or stress conditions.

• Protein-protein interactions: Interaction with regulatory proteins might sequester or activate glucokinase depending on metabolic needs.

• Redox regulation: Given S. maltophilia's ability to adapt to various environments, its glucokinase might contain cysteine residues subject to redox regulation, allowing activity modulation based on oxidative stress conditions.

• Metal ion dependency: Activity regulation through binding of divalent cations (Mg2+, Mn2+) that are essential for catalysis but may vary in availability under different growth conditions.

Investigating these regulatory mechanisms requires approaches such as:

• Mass spectrometry to identify post-translational modifications

• Protein-protein interaction studies (pull-down assays, co-immunoprecipitation)

• Site-directed mutagenesis of potential regulatory sites

• Activity assays under varying redox conditions and metal ion concentrations

How can CRISPR-Cas9 genome editing be optimized for studying glucokinase function in S. maltophilia?

Developing CRISPR-Cas9 genome editing protocols for S. maltophilia requires addressing several technical challenges:

• Delivery systems: Given that S. maltophilia is known for antibiotic resistance and potential barriers to transformation, effective delivery systems need optimization:

  • Electroporation protocols with specific voltage and resistance settings

  • Triparental mating approaches similar to those used for mutagenesis in S. maltophilia K279a

  • Potentially adapting the pGPI-SceI/pDAI-SceI-SacB system that has been successful for unmarked deletion mutants in S. maltophilia

• Guide RNA design considerations:

  • S. maltophilia has a relatively high GC content, requiring careful sgRNA design to avoid off-target effects

  • PAM site availability analysis in the glk gene region

  • Testing multiple sgRNAs targeting different regions of the gene

• Homology-directed repair templates:

  • Using longer homology arms (1-2 kb) to increase recombination efficiency

  • Including selectable markers flanked by FRT sites for subsequent removal

  • Designing precise mutations to study specific aspects of glucokinase function

• Verification approaches:

  • PCR screening of transformants

  • Sequencing confirmation of edited regions

  • Enzyme activity assays to confirm functional consequences

  • Whole genome sequencing to check for off-target effects

This approach builds upon successful genetic manipulation techniques used in S. maltophilia, such as the generation of the K279a Δrml mutant defective in O-side chain biosynthesis, which employed similar genomic modification strategies .

How does S. maltophilia glucokinase compare with the enzyme in closely related species like Xanthomonas?

S. maltophilia glucokinase likely shares structural and functional similarities with homologous enzymes in related species like Xanthomonas, but with specific adaptations reflecting ecological niche differences:

• Evolutionary relationships: S. maltophilia shows phylogenetic relatedness to Xanthomonas, as evidenced by the compatibility of their Type IV secretion systems. Studies have shown that expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating functional conservation between these related genera .

• Structural conservation and divergence: While core catalytic domains are likely conserved, specific substrate binding residues may show adaptations reflecting the different carbon source preferences of these bacteria. S. maltophilia can utilize uncommon carbon sources like L-glucitol through specialized enzyme systems , suggesting potential specialization in its glucokinase compared to Xanthomonas species.

• Regulatory differences: The regulation of glucose metabolism genes likely differs between these genera based on their distinct ecological niches—S. maltophilia as an opportunistic pathogen in humans and Xanthomonas as primarily plant pathogens.

• Expression patterns: In S. maltophilia, studies show that glucose-grown cells produce a different set of enzymes compared to cells grown on other carbon sources, indicating sophisticated carbon source-dependent regulation that may differ from Xanthomonas species .

Comparative analysis using sequence alignment, homology modeling, and enzyme kinetics would provide valuable insights into how glucokinase has evolved differently in these related but ecologically distinct bacterial genera.

What can we learn from structural studies of glucokinase across different bacterial species that informs S. maltophilia research?

Structural studies of bacterial glucokinases offer valuable insights for S. maltophilia research:

• Conservation of catalytic mechanism: Structural studies of bacterial glucokinases typically reveal a conserved ATP-binding pocket and glucose-binding site, suggesting similar catalytic mechanisms across species that likely apply to S. maltophilia glucokinase.

• Species-specific substrate binding adaptations: Subtle differences in the glucose-binding pocket can explain substrate preference variations. For S. maltophilia, which shows adaptability to various carbon sources , structural studies might reveal a more flexible substrate-binding site compared to more specialized bacteria.

• Regulatory site identification: Structures of bacterial glucokinases sometimes reveal allosteric binding sites or regions involved in protein-protein interactions that regulate activity. Identifying these in S. maltophilia glucokinase could explain its role in the organism's metabolic flexibility.

• Rational design opportunities: Structural information enables structure-based drug design targeting S. maltophilia glucokinase or rational enzyme engineering for biotechnological applications.

• Evolution of specificity determinants: Comparative structural analysis can identify the specific amino acid changes that have occurred during evolution to adapt glucokinase function to different bacterial metabolic requirements and ecological niches.

These structural insights are particularly relevant given S. maltophilia's clinical importance as an opportunistic pathogen with intrinsic antimicrobial resistance , as they may reveal potential metabolic vulnerabilities that could be targeted therapeutically.

How might recombinant S. maltophilia glucokinase be utilized in biosensor development for glucose detection?

Recombinant S. maltophilia glucokinase offers several advantages for biosensor development:

• Coupling with detection systems: The glucokinase-catalyzed ATP-dependent phosphorylation of glucose can be coupled to various detection methods:

  • Electrochemical detection of ADP production

  • Optical methods tracking NADPH generation via coupled enzyme systems

  • ATP consumption monitoring via luciferase-based systems

• Potential advantages over other glucose-detecting enzymes:

  • Unlike glucose oxidase, glucokinase doesn't produce hydrogen peroxide, potentially reducing interference in complex samples

  • May offer different substrate specificity profiles compared to widely-used glucose-monitoring enzymes

  • The ATP-dependent reaction can be leveraged for novel detection strategies

• Immobilization strategies:

  • Covalent attachment to functionalized surfaces using cross-linking reagents

  • Encapsulation in polymeric matrices that maintain enzyme activity while providing physical stability

  • Site-directed mutagenesis to introduce specific residues for oriented immobilization

• Performance optimization:

  • Engineering for improved stability at room temperature

  • Modifying substrate specificity through rational design based on structural information

  • Enhancing operational stability through protein engineering

Given S. maltophilia's adaptation to diverse environments , its glucokinase might offer unique properties like tolerance to interfering substances or stability under challenging conditions that could be advantageous for specialized biosensor applications.

What are the most promising approaches for inhibitor development targeting S. maltophilia glucokinase for potential antimicrobial applications?

Developing inhibitors targeting S. maltophilia glucokinase represents a potential novel antimicrobial strategy:

• Structure-based design approaches:

  • Virtual screening against the ATP-binding site, which likely has structural differences from human hexokinases

  • Fragment-based drug discovery focusing on unique pockets in the bacterial enzyme

  • Rational design of transition-state analogs specific to the bacterial catalytic mechanism

• Allosteric inhibitor development:

  • Targeting regulatory sites unique to bacterial glucokinases

  • Designing molecules that lock the enzyme in inactive conformations

  • Disrupting protein-protein interactions essential for enzyme function

• Targeting for biofilm prevention:

  • S. maltophilia biofilm formation capabilities vary among strains and may be influenced by glucose metabolism

  • Glucokinase inhibitors could potentially reduce biofilm formation by limiting the carbon flux through central metabolism

• Synergistic approaches:

  • Combining glucokinase inhibition with existing antibiotics to overcome resistance

  • Dual targeting of multiple enzymes in glucose metabolism to prevent metabolic bypass

  • Leveraging glucokinase inhibition to enhance the activity of antibiotics targeting cell wall synthesis

The development of such inhibitors must consider S. maltophilia's intrinsic resistance mechanisms, including efflux pumps and β-lactamases, which contribute to its high resistance rates to various antibiotics (30.5% resistance to trimethoprim-sulfamethoxazole and levofloxacin) .

What emerging technologies could advance our understanding of S. maltophilia glucokinase's role in metabolic adaptation to different environments?

Several cutting-edge technologies show promise for elucidating S. maltophilia glucokinase's role in metabolic adaptation:

• Single-cell metabolomics:

  • Tracking glucose metabolism at the single-cell level within heterogeneous S. maltophilia populations

  • Correlating metabolic profiles with gene expression and antibiotic susceptibility

  • Investigating metabolic adaptation during host interaction or biofilm formation

• Cryo-electron microscopy (Cryo-EM):

  • Determining high-resolution structures of S. maltophilia glucokinase in different functional states

  • Visualizing conformational changes during catalysis

  • Elucidating interactions with regulatory proteins or other metabolic enzymes

• CRISPR interference (CRISPRi) for fine-tuned regulation:

  • Precise modulation of glucokinase expression levels without complete gene knockout

  • Studying dose-dependent effects of glucokinase activity on metabolism and virulence

  • Temporal control of expression to study adaptation dynamics

• Metabolic flux analysis using stable isotopes:

  • 13C-labeled glucose tracking to quantify flux through different pathways

  • Comparing flux distributions between clinical and environmental isolates from different phylogenetic clades

  • Measuring changes in metabolic flux under different growth conditions or stresses

• In vivo imaging of metabolic dynamics:

  • Fluorescent biosensors for real-time tracking of glucose-6-phosphate levels

  • Visualizing metabolic changes during host-pathogen interactions

  • Spatial resolution of metabolism in biofilm structures