Recombinant Escherichia coli O45:K1 Glucokinase (glk)

What is Glucokinase (glk) and what is its role in E. coli metabolism?

Glucokinase (Glk) is an ATP-dependent enzyme in E. coli that phosphorylates glucose to produce glucose-6-phosphate, representing a critical step in glucose metabolism. Unlike hexokinases, glucokinases exhibit narrow specificity for glucose as a substrate. In E. coli, glucokinase processes intracellular glucose imported via phosphoenolpyruvate-dependent phosphotransferase system-independent uptake pathways. The enzyme catalyzes the addition of a phosphate group from ATP to the 6-position of glucose, producing glucose-6-phosphate which subsequently enters glycolysis or other metabolic pathways .

What distinguishes E. coli O45:K1 from other E. coli strains, and why is it significant for research?

E. coli O45:K1 represents a highly pathogenic clone that has emerged in France as a cause of neonatal meningitis. The O45 antigen is unusual in extraintestinal pathogenic E. coli (ExPEC) strains but has been identified in this virulent clone. The strain's significance lies in its combination of the O45 somatic antigen with the K1 capsular antigen, a combination associated with enhanced virulence, particularly in causing meningitis . The O45:K1:H7 serotype is closely related to the globally distributed archetypal clone O18:K1:H7 but differs in its O-antigen structure, making it an important subject for research into bacterial virulence evolution .

How does the O45 antigen potentially influence glucokinase function in pathogenic E. coli strains?

While direct evidence for O45 antigen regulation of glucokinase expression is limited, several indirect mechanisms may exist. The unique O45 antigen structure could alter cell surface properties, potentially affecting nutrient uptake patterns and consequently the regulation of metabolic enzymes like glucokinase . Additionally, pathogenic E. coli strains often coordinate virulence factor expression with metabolic changes. The O45 antigen gene cluster in strain S88 appears to have been acquired through horizontal gene transfer, potentially bringing along regulatory elements that could affect nearby genes or global regulatory networks . The relationship between O-antigen expression and central metabolism represents an important area for further investigation in these pathogenic strains.

What is the structural organization of E. coli Glucokinase and how does it compare to other sugar kinases?

E. coli Glucokinase (Glk) is a homodimer composed of two identical 321 amino acid subunits. Each monomer folds into two distinct domains: a small α/β domain (residues 2-110 and 301-321) and a larger α+β domain (residues 111-300) . The active site is located in a deep cleft between these two domains. Structural studies reveal that E. coli Glk shares structural similarities with Saccharomyces cerevisiae hexokinase and human brain hexokinase I, despite limited sequence homology . When glucose binds, it induces a conformational change resulting in closure of the small domains, with a maximal Cα shift of approximately 10Å. This "induced fit" mechanism positions the substrate optimally for phosphoryl transfer .

What is the catalytic mechanism of E. coli Glucokinase, and which residues are critical for its function?

The catalytic mechanism of E. coli Glucokinase involves several key steps, beginning with glucose binding in the active site cleft between the two domains, followed by a conformational change upon glucose binding. Critical residues in this process include Asp100, which functions as the general base that abstracts a proton from the O6 hydroxyl of glucose, enabling nucleophilic attack at the γ-phosphoryl group of ATP . Additional important residues include Asn99, Glu157, His160, and Glu187, which form hydrogen bonds with glucose and help position it correctly in the active site . The bound glucose forms hydrogen bonds with these residues, most of which (except His160) are structurally conserved in human hexokinase 1, suggesting a conserved glucose-binding mechanism despite evolutionary divergence .

What is the complete amino acid sequence of recombinant E. coli Glucokinase, and how is it typically expressed?

The complete amino acid sequence of recombinant E. coli Glucokinase typically includes a His-tag for purification purposes. The sequence is:

MGSSHHHHHHSSGLVPRGSHM GSMTKYALVGDVGGTNARLAL CDIASGEISQAKTYSGLDY PSLEAVIRVYLEEHKVEVKD GCIAIACPITGDWVAMTNH TWAFSIAEKKKNLGFSHLEI INDFTAVSMAIMLKKEHLIQ FGGAEPVEGKPIAIVGAGT GLGVAHLVHVDKRWVSLPGE GGHVDFAPNSEEAIILEILR AEIGHVSAERVLSGPGLVNL YRAIVKADNRLPENLKPKDI TERA

The protein is typically expressed in E. coli expression systems, with a molecular weight of approximately 35 kDa and a purity of >95% when analyzed by SDS-PAGE . The recombinant protein maintains the enzymatic activity of native glucokinase while providing additional features that facilitate purification and experimental manipulation.

What are the optimal conditions for expressing and purifying recombinant E. coli O45:K1 Glucokinase?

Optimal expression and purification of recombinant E. coli O45:K1 Glucokinase involves several critical parameters:

Expression Conditions:

• Host strain: BL21(DE3) or similar strains with reduced protease activity

• Growth medium: Rich media (LB or 2xYT) for high yield

• Temperature: 18-25°C post-induction to enhance proper folding

• IPTG concentration: 0.1-0.5 mM, with lower concentrations often yielding more soluble protein

• Induction time: 16-20 hours at lower temperatures

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT

• Affinity chromatography using Ni-NTA resin for His-tagged protein

• Size exclusion chromatography to separate dimeric active enzyme from aggregates

• Storage in buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 10% glycerol

The purified enzyme should be flash-frozen and stored at -80°C for long-term stability, where it typically retains >90% activity for at least 6 months .

What assays are most reliable for measuring E. coli Glucokinase activity, and what are their limitations?

Several assays can be used to measure E. coli Glucokinase activity, each with specific advantages and limitations:

1. Coupled Spectrophotometric Assay:

• Method: Links glucokinase activity to NADH production/consumption through auxiliary enzymes (glucose-6-phosphate dehydrogenase)

• Advantages: Continuous, real-time monitoring; high sensitivity

• Limitations: Auxiliary enzymes can be limiting factors; potential for interference from other ATP-consuming enzymes

2. ADP Production Assay:

• Method: Measures ADP produced during the reaction using ADP-specific detection reagents

• Advantages: Direct measurement of reaction product; compatible with high-throughput screening

• Limitations: End-point measurement; generally more expensive

Comparison of Key Parameters:

When selecting an assay, researchers should consider their specific experimental needs, available equipment, and the nature of their samples (pure enzyme vs. cell lysates) .

How can site-directed mutagenesis be used to study critical residues in E. coli O45:K1 Glucokinase?

Site-directed mutagenesis provides a powerful approach for investigating structure-function relationships in E. coli O45:K1 Glucokinase. A methodological framework includes:

Target Selection:

• Catalytic residues: Asp100 (catalytic base), Asn99, Glu157, His160, Glu187 (glucose binding)

• Domain interface residues: Those involved in domain closure upon substrate binding

• Dimer interface residues: To investigate the importance of dimerization

Mutation Strategies:

• Conservative substitutions: Replace with similar amino acids (e.g., Asp→Glu)

• Non-conservative substitutions: Dramatic changes to demonstrate essential nature (e.g., Asp→Ala)

• Function-swapping: Replace with residues found in related enzymes with different specificities

Functional Analysis:

• Expression and purification: Compare yields of mutant vs. wild-type

• Structural integrity: Circular dichroism spectroscopy, thermal stability assays

• Kinetic analysis: Determine Km, kcat, and kcat/Km for glucose and ATP

• Binding studies: Isothermal titration calorimetry (ITC) for substrate binding affinities

This approach allows researchers to create a detailed map of structure-function relationships in glucokinase and potentially identify residues that could be targeted for inhibitor design or protein engineering applications .

How can recombinant E. coli O45:K1 Glucokinase be used to study pathogenesis mechanisms in meningitis-causing strains?

Recombinant E. coli O45:K1 Glucokinase can serve as a valuable tool for investigating pathogenesis mechanisms in meningitis-causing strains through several research approaches:

Metabolic Adaptation During Infection:

• Compare glucokinase activity and expression levels between pathogenic O45:K1 strains and non-pathogenic E. coli

• Investigate how glucose metabolism shifts during different stages of infection

• Examine how glucokinase activity changes in response to host environments (blood, cerebrospinal fluid)

Virulence-Metabolism Coupling:

• Create glucokinase knockout or regulated expression strains in O45:K1 background

• Assess how altered glucose metabolism affects expression of virulence factors

• Determine if metabolic shifts governed by glucokinase activity correlate with virulence gene expression

In Vivo Infection Models:

• Use the neonatal rat meningitis model to assess how glucokinase mutations affect virulence

• Compare wild-type and glucokinase-modified strains for their ability to survive in bloodstream, cross the blood-brain barrier, and proliferate in cerebrospinal fluid

Such studies could reveal whether metabolic adaptations mediated by glucokinase contribute to the enhanced virulence of the O45:K1 clone and potentially identify new therapeutic targets .

What insights can comparative analysis provide about E. coli O45:K1 Glucokinase evolution in the context of pathogenic strains?

Comparative analysis of glucokinase from E. coli O45:K1 and other strains can reveal important evolutionary insights:

Phylogenetic Analysis:

• Compare glucokinase sequences between O45:K1 strains and other pathogenic/non-pathogenic E. coli

• Analyze the evolutionary relationship between glucokinase and the acquisition of virulence factors

• Examine potential horizontal gene transfer events that might have shaped glucokinase evolution

Structure-Function Relationships:

• Map strain-specific amino acid substitutions onto structural models

• Identify positively selected residues that might contribute to strain-specific adaptations

• Correlate structural differences with enzymatic properties

This approach is particularly important given that the O45 antigen gene cluster in pathogenic strains appears to have been acquired through horizontal gene transfer events . Understanding whether glucokinase has co-evolved with virulence determinants could provide insights into the metabolic adaptation of emerging pathogenic clones.

How do specific mutations in glucokinase affect E. coli O45:K1 virulence in experimental infection models?

The relationship between glucokinase mutations and virulence can be systematically investigated using experimental infection models. Research approaches include:

Mutation Design Strategy:

• Create specific point mutations affecting catalytic efficiency

• Generate domain-swapped variants between pathogenic and non-pathogenic strains

• Develop conditional expression systems to control glucokinase activity during infection

Infection Model Selection:

• Neonatal rat meningitis model for studying E. coli O45:K1 pathogenesis

• Cell culture models of blood-brain barrier penetration

• Ex vivo cerebrospinal fluid survival assays

Key Parameters to Measure:

• Bacterial load in blood and cerebrospinal fluid

• Inflammatory response markers

• Host survival rates

• Transcriptional changes in both host and pathogen

Studies in the neonatal rat meningitis model have already demonstrated the crucial role of the O45 antigen in virulence . Extending this approach to investigate glucokinase mutations could reveal whether central metabolism plays a direct role in pathogenesis or primarily supports virulence factor expression.

What are common challenges in expressing recombinant E. coli O45:K1 Glucokinase, and how can they be addressed?

Researchers often encounter several challenges when expressing recombinant E. coli O45:K1 Glucokinase:

Poor Solubility and Inclusion Body Formation:

• Problem: Overexpression can lead to protein aggregation

• Solutions: Reduce induction temperature to 16-18°C; decrease IPTG concentration to 0.1-0.2 mM; co-express with chaperones; use solubility-enhancing fusion tags; add osmolytes to culture medium

Low Enzymatic Activity:

• Problem: Recombinant enzyme shows significantly lower activity than expected

• Solutions: Ensure proper folding by optimizing purification conditions; add stabilizing agents (DTT, glycerol); include glucose in purification buffers; verify protein integrity

Proteolytic Degradation:

• Problem: The enzyme shows degradation bands on SDS-PAGE

• Solutions: Use protease-deficient host strains; include protease inhibitors; perform purification steps at 4°C; add EDTA to inhibit metalloproteases

By systematically addressing these challenges, researchers can significantly improve the expression and purification of functional recombinant E. coli O45:K1 Glucokinase .

What are the key considerations for designing kinetic studies with recombinant E. coli O45:K1 Glucokinase?

Designing rigorous kinetic studies with recombinant E. coli O45:K1 Glucokinase requires careful consideration of multiple experimental parameters:

Enzyme Preparation Considerations:

• Purity requirement: >95% as verified by SDS-PAGE

• Activity verification before kinetic studies

• Storage conditions: Aliquot and store at -80°C; avoid multiple freeze-thaw cycles

• Buffer composition: 20-50 mM Tris or HEPES, pH 7.5-8.0, 50-100 mM NaCl, 5 mM MgCl₂, 1 mM DTT

Substrate Considerations:

• For Glucose: Concentration range 0.1-10× Km (typical Km ~0.2-0.5 mM)

• For ATP: Concentration range 0.1-10× Km (typical Km ~0.3-0.7 mM)

• Include Mg²⁺ (typically 5-10 mM) as ATP·Mg complex is the true substrate

Experimental Design for Kinetic Parameter Determination:

• Ensure <10% substrate consumption (linearity of progress curves)

• Include sufficient time points for reliable rate determination

• Use appropriate enzyme concentration (typically 10-100 nM)

• Design experiments to distinguish between different kinetic mechanisms

Following these guidelines enables researchers to design robust kinetic experiments that yield reliable mechanistic insights into the catalytic behavior of E. coli O45:K1 Glucokinase .

How can researchers effectively compare enzymatic properties of glucokinase from different E. coli strains in virulence studies?

Effective comparison of glucokinase enzymatic properties from different E. coli strains requires a systematic approach:

Standardized Cloning and Expression Strategy:

• Use identical vector backbones, tags, and cloning sites

• Express all variants under identical conditions

• Purify using the same protocol and buffer systems

• Verify comparable purity and proper folding

Comparative Kinetic Analysis:

• Determine Km, kcat, and kcat/Km for both glucose and ATP

• Measure substrate specificity profiles

• Analyze pH-activity profiles and temperature optima

Example Data Presentation Format:

Integration with Virulence Studies:

• Correlate enzymatic parameters with virulence metrics

• Use gene replacements to swap glucokinase variants between strains

• Measure impact on fitness in infection models

By applying this comprehensive comparative framework, researchers can identify subtle but potentially important differences in glucokinase properties between pathogenic and non-pathogenic E. coli strains, potentially revealing adaptations that contribute to virulence or host adaptation .