Recombinant Neisseria gonorrhoeae Glucokinase (glk)

What is the evolutionary conservation of glucokinase across Neisseria species?

The glucokinase (glk) gene in Neisseria gonorrhoeae shows high conservation with closely related bacteria. As observed with other genes in the Neisseria genus, there is an identical gene arrangement present in Neisseria gonorrhoeae (strain FA 1090) and Neisseria lactamica (ST-640) . This conservation suggests evolutionary importance of glucokinase in basic metabolic functions across Neisseria species.

Studies on other bacterial transporters and metabolic enzymes within the Neisseria family indicate that while core catalytic domains maintain high conservation, regulatory regions may show greater variability, reflecting adaptation to different host environments and metabolic needs.

How does N. gonorrhoeae glucokinase differ functionally from human glucokinase?

N. gonorrhoeae glucokinase and human glucokinase (GCK) represent distinct evolutionary branches with significant functional differences:

• Regulatory mechanisms: Human GCK displays positive cooperativity despite being monomeric and possessing only a single glucose binding site , whereas bacterial glucokinases typically follow Michaelis-Menten kinetics without cooperativity.

• Structural organization: Human GCK contains a small domain that undergoes intrinsic disorder-order transitions critical for its glucose-sensing function . This mechanism involves millisecond timescale order-disorder transitions that govern cooperativity and allostery . Bacterial glucokinases generally maintain more rigid structures without these extensive conformational changes.

• Substrate affinity: Bacterial glucokinases typically have different glucose affinities compared to human GCK, which has evolved specifically for glucose sensing at physiological concentrations in blood.

These differences make bacterial glucokinases potentially valuable targets for antimicrobial development with minimal host enzyme interference.

What expression systems are most effective for producing active recombinant N. gonorrhoeae glucokinase?

For recombinant expression of N. gonorrhoeae glucokinase, E. coli-based systems have proven most effective, particularly when optimized for bacterial protein expression. Drawing from approaches used with other bacterial enzymes:

• Expression vector selection: pET-based vectors under T7 promoter control provide high-level expression. GST-fusion systems have been successfully employed for other glucokinases, as demonstrated in studies where "recombinant enzyme [was produced] in Escherichia coli" with good yields .

• Host strain optimization: E. coli BL21(DE3) derivatives are recommended, particularly Rosetta strains if N. gonorrhoeae codon usage differs significantly from E. coli.

• Induction conditions: Lower temperatures (16-25°C) often improve folding of active enzyme, with extended expression times (16-24 hours) at reduced IPTG concentrations (0.1-0.5 mM).

• Solubility enhancement: Co-expression with chaperones (GroEL/GroES) may increase soluble protein yield if inclusion body formation occurs.

Methodologically, optimizing these parameters through small-scale expression trials, with activity measurements at each step, is essential before scaling up production.

What are the critical factors for maintaining enzyme activity during purification of recombinant N. gonorrhoeae glucokinase?

Several critical factors must be controlled to maintain activity during purification:

• Buffer composition:

  • pH maintenance (typically 7.0-8.0)

  • Inclusion of stabilizing agents:

    • Glycerol (10-20%)

    • Reducing agents (1-5 mM DTT or β-mercaptoethanol)

    • Divalent cations (Mg²⁺ at 1-5 mM) as cofactors

• Purification strategy:

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Intermediate purification via ion exchange chromatography

  • Final polishing by size exclusion chromatography

• Temperature control: Maintain all purification steps at 4°C and avoid freeze-thaw cycles

• Activity assay development: Implement a reliable activity assay at each purification step to track specific activity

In developing these protocols, researchers should note that studies with human glucokinase mutants demonstrated that thermal shift unfolding assays using tryptophan fluorescence (I320/360) can help identify conditions that preserve structural integrity , a technique that could be adapted for N. gonorrhoeae glucokinase.

How should kinetic parameters of recombinant N. gonorrhoeae glucokinase be determined?

Determining accurate kinetic parameters for recombinant N. gonorrhoeae glucokinase requires systematic methodology:

• Coupled enzyme assay approach:

  • Primary reaction: Glucose + ATP → Glucose-6-phosphate + ADP (catalyzed by glucokinase)

  • Coupling reaction: Glucose-6-phosphate + NADP⁺ → 6-phosphogluconate + NADPH (catalyzed by G6P dehydrogenase)

  • Measurement: Spectrophotometric monitoring of NADPH formation at 340 nm

• Direct ADP formation assay:

  • Use of ADP-Glo™ or similar luminescence-based ADP detection systems

  • Provides higher sensitivity for low-activity preparations

• Experimental design for parameter determination:

  • Variable glucose concentrations (0.05-50 mM) at fixed ATP (typically 2-5 mM)

  • Variable ATP concentrations (0.01-10 mM) at fixed glucose (typically at saturating levels)

  • Temperature optimization (typically 25-37°C)

  • pH profiling (pH 6.0-9.0)

• Data analysis:

  • Use of non-linear regression for Michaelis-Menten parameters (Km, Vmax, kcat)

  • Evaluation of alternative kinetic models if deviation from Michaelis-Menten is observed

Drawing from studies of human glucokinase, it's important to determine the presence or absence of cooperativity. Human GCK shows positive cooperativity despite having only one glucose binding site , while bacterial glucokinases typically don't exhibit this property.

What experimental approaches can identify potential allosteric regulators of N. gonorrhoeae glucokinase?

Identifying allosteric regulators requires multiple complementary approaches:

• Thermal shift assays:

  • Monitor protein unfolding temperatures (Tm) in the presence of candidate molecules

  • Significant Tm shifts indicate binding and potential structural stabilization

  • Tryptophan fluorescence (I320/360) has proven valuable for thermal shift analysis of glucokinases

• Activity modulators screening:

  • Systematic testing of metabolic intermediates (particularly TCA cycle components)

  • Assessment of nucleotides beyond ATP (GTP, UTP)

  • Evaluation of small molecules from host environments

• Structural approaches:

  • NMR spectroscopy using isotopically labeled enzyme (¹³C-Ile or ¹⁵N labeling)

  • X-ray crystallography with and without potential modulators

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered dynamics

• Mutational analysis:

  • Strategic mutations in putative allosteric sites

  • Domain-swapping experiments with related enzymes

Research on human glucokinase has revealed that it contains distinct activation mechanisms, classified as α-type activation (where glucose affinity increases) and β-type activation (where glucose affinity remains largely unchanged) . Similar diverse regulatory mechanisms may exist in bacterial glucokinases and require systematic investigation.

What structural features of N. gonorrhoeae glucokinase are critical for catalysis?

Based on broader glucokinase research, several structural features are likely critical for N. gonorrhoeae glucokinase catalysis:

• Active site architecture:

  • Glucose binding pocket with specific hydrogen-bonding residues

  • ATP binding cassette with magnesium coordination sites

  • Catalytic residues for phosphoryl transfer

• Domain organization:

  • Large domain (containing most of the active site)

  • Small domain (providing dynamic elements for substrate binding)

  • Interdomain flexibility that permits induced-fit conformational changes

• Functionally critical regions:

  • Glycine-rich loop for ATP binding (similar to P-loop in kinases)

  • Glucose specificity loop

  • Catalytic base (typically aspartic acid)

Studies of human glucokinase mutations have demonstrated that alterations in the ATP binding cassette can significantly impact catalytic efficiency without necessarily affecting glucose binding . For instance, the P417R mutation in human glucokinase affects activity significantly while preserving glucose recognition . Similar structure-function relationships may exist in N. gonorrhoeae glucokinase.

How can protein engineering be employed to enhance stability of recombinant N. gonorrhoeae glucokinase?

Protein engineering strategies to enhance stability can be implemented through:

• Rational design approaches:

  • Introduction of disulfide bridges at positions identified through computational modeling

  • Surface charge optimization to reduce aggregation potential

  • Proline substitutions in loop regions to reduce flexibility and proteolytic susceptibility

  • Elimination of proteolytically susceptible sites identified by limited proteolysis experiments

• Directed evolution methods:

  • Error-prone PCR to generate mutation libraries

  • DNA shuffling with glucokinases from thermophilic bacteria

  • Selection systems in glucokinase-deficient bacterial strains (similar to approaches used for human GCK, where "positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium" proved successful)

• Consensus approach:

  • Analysis of glucokinase sequences from multiple Neisseria species

  • Introduction of consensus residues at divergent positions

• Chimeric enzyme development:

  • Domain swapping with more stable glucokinases from related bacteria

  • Introduction of thermostable enzyme elements while preserving catalytic function

Studies of human glucokinase have revealed that thermostability correlates with severity of hyperglycemia in mutants , suggesting that thermal stability is tightly linked to proper enzyme function. Similar relationships may exist for bacterial glucokinases, making stability engineering an important research direction.

How do the catalytic properties of N. gonorrhoeae glucokinase compare with those from other pathogenic bacteria?

A comparative analysis reveals distinctive characteristics:

*Estimated based on typical bacterial glucokinases and related Neisseria species enzymes

The catalytic efficiency (kcat/Km) of bacterial glucokinases differs substantially from human GCK. Human glucokinase has a high Km for glucose (5-10 mM) and significant cooperativity, while bacterial enzymes typically have lower Km values and follow Michaelis-Menten kinetics. In some cases, engineered human GCK variants have shown "a 100-fold increase in catalytic efficiency over wild-type GCK" , demonstrating the substantial functional plasticity of these enzymes.

What can be learned from glucokinase mutations in other organisms that might apply to studying N. gonorrhoeae glucokinase?

Extensive research on glucokinase mutations provides valuable insights for N. gonorrhoeae studies:

• Functionally critical residues:

  • Mutations affecting glucose binding (e.g., D205H in human GCK) severely impair enzyme function

  • ATP binding site mutations (e.g., G246V, H416R in human GCK) alter kinetic parameters

  • Mutations in the allosteric activator site (e.g., A449T in human GCK) can dramatically modify kinetic parameters

• Structural insights:

  • Research has identified that human GCK contains a 30-residue active-site loop that closes upon glucose binding

  • Limited proteolysis has been effective in mapping disorder in unliganded enzyme

  • NMR studies using ¹³C-labeled isoleucine methyl groups have revealed important conformational dynamics

• Engineering possibilities:

  • Some mutations can lead to hyperactive variants with reduced cooperativity (relevant to bacterial enzymes lacking cooperativity)

  • Structure-guided mutations can enhance thermostability

  • Regulatory site modifications can alter allosteric control

These findings suggest strategies for investigating structure-function relationships in N. gonorrhoeae glucokinase and potentially engineering variants with desired properties for research or biotechnological applications.

What experimental approaches can evaluate N. gonorrhoeae glucokinase as a potential antimicrobial target?

Comprehensive evaluation requires multi-faceted approaches:

• Essentiality determination:

  • CRISPR interference or gene deletion studies in N. gonorrhoeae

  • Growth studies with carbon sources requiring glucokinase activity

  • Complementation experiments to confirm phenotypes

• Inhibitor development pipeline:

  • High-throughput screening assays using purified recombinant enzyme

  • Fragment-based lead discovery targeting unique structural features

  • Structure-guided drug design based on crystallographic data

  • Computational screening focused on bacterial-specific binding sites

• Selectivity assessment:

  • Counter-screening against human hexokinases and glucokinase

  • Analysis of inhibition mechanisms (competitive, non-competitive, uncompetitive)

  • Determination of inhibition constants (Ki) and IC₅₀ values

• Cellular activity evaluation:

  • Minimum inhibitory concentration (MIC) determination

  • Time-kill kinetics

  • Resistance development assessment through serial passage

Drawing from experiences with other metabolic enzyme targets, the most promising approach may involve targeting bacterial-specific regulatory sites rather than the highly conserved catalytic core.

How can N. gonorrhoeae glucokinase activity be assessed in intracellular environments during infection?

Assessing activity during infection requires specialized methodologies:

• Reporter systems:

  • Development of transcriptional fusions between glk promoter and reporters (GFP, luciferase)

  • Translational fusions maintaining enzymatic activity with minimal tags

  • Biosensors detecting glucose-6-phosphate levels

• Infection models:

  • Cell invasion and intracellular persistence assays similar to those developed for studying the l-glutamate ABC transporter in N. meningitidis

  • Expression analysis during cell invasion to assess regulation

  • Ex vivo tissue models that recapitulate native infection environments

• Metabolic analysis:

  • ¹³C-glucose labeling to track metabolic flux through glycolysis

  • Metabolomics profiling of infected versus uninfected cells

  • Comparison of wild-type and glucokinase-deficient strains

• Direct activity measurements:

  • Glucose consumption rates in infection models

  • ATP/ADP ratios as indicators of metabolic activity

  • Selective permeabilization of host cells to assay enzyme activity

Studies on N. meningitidis have shown that expression data during cell invasion can provide critical insights into adaptation mechanisms during infection , suggesting similar approaches would be valuable for N. gonorrhoeae glucokinase research.

What are the common challenges in obtaining active recombinant N. gonorrhoeae glucokinase and how can they be addressed?

Researchers frequently encounter specific challenges when working with recombinant bacterial glucokinases:

• Low expression yield:

  • Optimize codon usage for expression host

  • Test multiple fusion tags (His₆, GST, MBP)

  • Evaluate different promoter systems (T7, tac, araBAD)

  • Screen multiple expression conditions (temperature, induction time, media composition)

• Protein insolubility:

  • Lower expression temperature (16-20°C)

  • Co-express with molecular chaperones

  • Use solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • Optimize lysis buffer composition (detergents, osmolytes)

• Loss of activity during purification:

  • Include stabilizing agents (glycerol, reducing agents)

  • Minimize purification steps and time

  • Maintain constant cofactor presence (Mg²⁺)

  • Avoid freeze-thaw cycles by preparing single-use aliquots

• Inconsistent activity measurements:

  • Standardize enzyme concentration determination methods

  • Validate activity assays with known standards

  • Control reaction components rigorously

  • Account for potential lag phases in coupled assays

The use of GST-fusion proteins has been successful for other glucokinases, as noted in studies where "recombinant enzyme was produced in Escherichia coli" , suggesting this approach may be valuable for N. gonorrhoeae glucokinase.

How can researchers distinguish between enzyme inactivity due to improper folding versus inhibition by buffer components?

Distinguishing these issues requires systematic troubleshooting:

• Structural analysis:

  • Circular dichroism spectroscopy to assess secondary structure

  • Tryptophan fluorescence to evaluate tertiary structure integrity

  • Size exclusion chromatography to detect aggregation

  • Thermal shift assays to determine stability in different buffers

• Activity recovery experiments:

  • Systematic buffer exchange via dialysis or gel filtration

  • Addition of potential activators or cofactors

  • Denaturation and refolding attempts

  • Testing of enzyme activity under various buffer conditions

• Inhibition analysis:

  • Individual testing of buffer components for inhibitory effects

  • Construction of inhibition curves for suspect components

  • Competition experiments with substrates

  • Comparison with control enzymes (e.g., yeast hexokinase)

• Protein quality assessment:

  • Mass spectrometry to confirm intact protein

  • Limited proteolysis to assess structural integrity

  • N-terminal sequencing to verify correct processing

  • Dynamic light scattering to evaluate homogeneity

Research on human glucokinase has shown that "disruption of the glucokinase regulatory protein-binding site (GCK⁽K140E⁾), but not the ATP binding cassette (GCK⁽P417R⁾), prevented inhibition of enzyme activity" , highlighting how specific structural regions can dramatically affect enzyme regulation and activity. Similar region-specific effects may occur in bacterial glucokinases.