Recombinant Klebsiella pneumoniae subsp. pneumoniae Serine hydroxymethyltransferase (glyA)

What is the primary biochemical function of Klebsiella pneumoniae glyA?

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, primarily catalyzes the reversible interconversion of L-serine to glycine by transferring a methyl group to tetrahydrofolate, producing 5,10-methylenetetrahydrofolate. This reaction represents a critical step in one-carbon metabolism essential for nucleotide synthesis and amino acid metabolism . The enzyme utilizes pyridoxal 5'-phosphate (PLP) as a cofactor, forming a Schiff base with a conserved lysine residue in the active site. Beyond its primary function, glyA demonstrates remarkable catalytic diversity, including decarboxylation, transamination, and retroaldol cleavage activities .

For experimental verification of K. pneumoniae glyA function, researchers should implement spectrophotometric assays monitoring glycine formation or use coupled enzyme systems that track the generation of 5,10-methylenetetrahydrofolate through fluorescence detection. When designing such assays, ensure inclusion of PLP (50-80 μM) and appropriate reducing agents to maintain enzyme stability.

How does glyA structure compare across bacterial species?

While the search results don't provide specific structural information for K. pneumoniae glyA, comparative analysis with characterized bacterial SHMTs reveals important insights. GlyA is highly conserved across bacterial species, including Escherichia coli, Chlamydia pneumoniae, and Tannerella forsythia . This conservation suggests structural similarities that underpin its essential metabolic functions.

The most thoroughly characterized bacterial SHMT is from E. coli, which functions as a homodimer with each subunit containing approximately 417 amino acids . For structural investigations of K. pneumoniae glyA, researchers should implement:

• Homology modeling based on crystallized bacterial SHMTs

• Secondary structure prediction using circular dichroism spectroscopy

• Thermal shift assays to assess structural stability under various conditions

• Site-directed mutagenesis of predicted catalytic residues followed by activity assays

Importantly, when preparing recombinant K. pneumoniae glyA for structural studies, inclusion of PLP in purification buffers is essential for maintaining structural integrity and enzymatic activity .

What pathways depend on glyA activity in bacterial metabolism?

The glyA enzyme occupies a central position in bacterial metabolism, participating in multiple essential pathways:

Beyond these primary pathways, research indicates that in some bacteria, glyA demonstrates alanine racemase activity, catalyzing the interconversion of L-alanine and D-alanine . This moonlighting function may be particularly significant in organisms lacking dedicated alanine racemases, as D-alanine is essential for peptidoglycan synthesis in bacterial cell walls.

To characterize the metabolic impact of glyA in K. pneumoniae specifically, researchers should implement metabolic flux analysis using isotope-labeled substrates (13C-serine) and track carbon flow through these interconnected pathways using LC-MS/MS techniques.

What expression systems are optimal for producing recombinant K. pneumoniae glyA?

Based on experimental protocols used for similar bacterial SHMTs, researchers should consider the following expression strategies for K. pneumoniae glyA:

For bacterial expression, E. coli BL21(DE3) or JM83 strains typically yield high levels of soluble protein when the gene is cloned into pET vectors (cytoplasmic expression) or pASK-IBA vectors (periplasmic expression with signal peptides) . The expression construct should include affinity tags such as His6 or Strep-tag for simplified purification.

Optimal expression conditions include:

• Growth medium: LB supplemented with 50 μM PLP and potentially 200 μM folinic acid

• Induction parameters: At OD600 of 1.0-1.2, induce with appropriate inducer (IPTG or anhydrotetracycline)

• Post-induction: Lower temperature (25°C) for 4-6 hours to enhance protein solubility

• Supplementation: Include 2 mM DTT to maintain reducing environment

For challenging expression cases, consider alternative approaches such as cell-free protein synthesis systems or baculovirus-mediated expression in insect cells, which may improve folding of complex proteins.

One critical consideration is ensuring PLP incorporation during expression. Researchers have demonstrated improved enzyme activity when expression media and purification buffers are supplemented with PLP (50-80 μM) .

What purification strategy yields the highest quality recombinant glyA enzyme?

A multi-step purification strategy is recommended for obtaining high-purity, catalytically active K. pneumoniae glyA:

• Initial Capture: Affinity chromatography using the fusion tag (Strep-Tactin for Strep-tagged proteins or Ni-NTA for His-tagged proteins)

• Intermediate Purification: Ion exchange chromatography to separate charge variants

• Polishing: Size exclusion chromatography to eliminate aggregates and ensure homogeneity

Throughout all purification steps, buffers should contain:

• PLP (50 μM) to maintain cofactor saturation

• Reducing agents (2 mM DTT) to prevent oxidation of catalytic cysteines

• Potentially low concentrations of detergents (0.1% N-lauroylsarcosine) for improved solubility

• Glycerol (10%) to enhance stability during storage

Quality control assessments should include SDS-PAGE, Western blotting, dynamic light scattering for aggregation analysis, and activity assays using the serine-to-glycine conversion reaction. Thermal shift assays can help optimize buffer conditions for maximum stability.

For storage, flash-freeze aliquots in liquid nitrogen and store at -80°C with 20-25% glycerol to prevent freeze-thaw damage. Avoid multiple freeze-thaw cycles as these significantly reduce enzymatic activity.

How can enzymatic activity of recombinant K. pneumoniae glyA be accurately measured?

Several complementary assay methods allow comprehensive characterization of K. pneumoniae glyA activity:

For the primary serine hydroxymethyltransferase activity:

• Spectrophotometric coupling assays measuring the formation of 5,10-methylenetetrahydrofolate

• Radiometric assays using 14C-labeled serine to quantify radiolabeled glycine production

• HPLC-based methods to directly quantify substrate consumption and product formation

For potential alanine racemase activity:

• D-amino acid oxidase (DAAO) coupled assay as described in the literature for C. pneumoniae GlyA

• Incubate glyA with L-alanine (50 mM) for 16 hours at 37°C in optimized buffer (50 mM KH2PO4, pH 8, 100 mM KCl, 80 μM PLP, 2 mM DTT)

• Detect D-alanine formation using DAAO to convert it to pyruvate

• Quantify pyruvate colorimetrically with 2,4-dinitrophenylhydrazine (DNPH)

Critical controls for all assays include:

• Enzyme-free reactions to account for non-enzymatic conversions

• Heat-inactivated enzyme controls

• Positive controls using commercially available SHMT or alanine racemase

• Inhibition controls using known inhibitors like D-cycloserine (10 mM)

Enzyme kinetic parameters (Km, kcat, kcat/Km) should be determined for each substrate under standardized conditions, providing a comprehensive catalytic profile of the recombinant enzyme.

How does glyA contribute to Klebsiella pneumoniae virulence?

While direct evidence for K. pneumoniae glyA's role in virulence is limited in the available search results, studies on the glyA gene in other bacterial pathogens provide valuable insights. In Tannerella forsythia, the glyA gene has been associated with virulence, playing a vital role in bacterial metabolism . Notably, research has shown that the frequency of the glyA gene was significantly higher (57.14%) in bacterial isolates from cases of aggressive periodontitis compared to milder forms of the disease .

To investigate K. pneumoniae glyA's contribution to virulence, researchers should implement:

• Gene knockout studies using CRISPR-Cas9 or homologous recombination to generate glyA deletion mutants

• Phenotypic characterization assessing growth rates, biofilm formation, and virulence factor production

• Infection models comparing wild-type and glyA-deficient strains in cell culture and animal models

• Transcriptomic analysis to identify genes whose expression changes in response to glyA perturbation

The enzyme's role in one-carbon metabolism suggests it may influence virulence through metabolic pathways that support rapid bacterial proliferation during infection. Additionally, if K. pneumoniae glyA possesses alanine racemase activity similar to C. pneumoniae GlyA , it could contribute to cell wall synthesis by providing D-alanine, potentially affecting cellular integrity and antibiotic susceptibility.

What methodologies effectively characterize moonlighting functions of K. pneumoniae glyA?

The search results indicate that SHMTs can perform multiple catalytic functions beyond their primary role in serine-glycine interconversion. Most notably, GlyA from C. pneumoniae demonstrates alanine racemase activity that can partially compensate for the absence of dedicated alanine racemases . This functional versatility presents interesting research opportunities but requires specialized methodologies:

For comprehensive functional characterization:

• Activity screening:

  • Systematically test purified recombinant glyA for various PLP-dependent reactions

  • Implement high-sensitivity assays capable of detecting minor activities

  • Use purified enzyme preparations to avoid interference from host enzymes

• Genetic complementation:

  • Express K. pneumoniae glyA in appropriate knockout strains (e.g., E. coli Δalr ΔdadX racemase double mutant)

  • Assess growth under conditions requiring the potentially complemented function

  • Measure growth rates with and without supplementation of relevant metabolites

• Structural analysis:

  • Perform crystallographic studies with different substrates or substrate analogs

  • Use molecular dynamics simulations to understand conformational flexibility

  • Identify structural elements that contribute to catalytic promiscuity

• Enzyme engineering:

  • Generate site-directed mutants targeting residues predicted to influence specific activities

  • Develop variants with enhanced or suppressed moonlighting functions

  • Create chimeric enzymes by domain swapping with other SHMTs

These approaches can reveal the full catalytic repertoire of K. pneumoniae glyA and provide insights into its potentially diverse roles in bacterial metabolism and pathogenesis.

How does environmental context affect glyA activity and expression?

Understanding how environmental factors influence glyA activity and expression is crucial for comprehending its role in bacterial adaptation and pathogenesis. While the search results don't directly address this for K. pneumoniae specifically, general principles and approaches can be outlined:

To investigate environmental regulation of glyA:

• Transcriptional regulation:

  • Implement qRT-PCR to measure glyA expression under varying conditions (pH, temperature, nutrient availability)

  • Use reporter gene fusions (glyA promoter-GFP) to monitor expression in real-time

  • Perform RNA-seq to identify co-regulated genes and regulatory networks

• Metabolic adaptation:

  • Analyze metabolomic profiles under conditions where glyA expression changes

  • Trace isotope-labeled metabolites to determine flux through glyA-dependent pathways

  • Compare intracellular folate pools and one-carbon metabolite levels across conditions

• Host-pathogen interface:

  • Examine glyA expression during infection of host cells

  • Determine how host-imposed stresses (nutritional immunity, oxidative stress) affect glyA function

  • Investigate glyA expression in biofilms versus planktonic cells

• Post-translational regulation:

  • Identify potential modifications that affect enzyme activity

  • Determine how redox conditions influence enzyme function

  • Assess protein-protein interactions that might modulate glyA activity

These investigations can reveal how K. pneumoniae adapts its central metabolism via glyA regulation to thrive in diverse environments, including those encountered during host infection.

How can inhibitor studies of K. pneumoniae glyA advance antimicrobial development?

The development of glyA inhibitors represents a promising approach for novel antimicrobial strategies. Based on information from the search results, we can outline methodologies for inhibitor studies and their potential applications:

From search result , we know that D-cycloserine (10 mM) can inhibit GlyA's alanine racemase activity. This provides a starting point for developing more specific and potent inhibitors targeting K. pneumoniae glyA.

Methodological approaches for inhibitor development:

• High-throughput screening:

  • Establish miniaturized activity assays suitable for screening compound libraries

  • Implement fluorescence-based detection methods for rapid evaluation

  • Screen natural product collections and synthetic chemical libraries

• Structure-based design:

  • Use homology models or crystal structures to identify binding pockets

  • Perform virtual screening against the enzyme's active site

  • Design transition-state analogs that exploit the PLP-dependent mechanism

• Fragment-based approaches:

  • Screen small chemical fragments for binding to glyA

  • Elaborate promising fragments into more potent inhibitors

  • Link fragments that bind to different sites to create high-affinity compounds

• Validation cascade:

  • Determine IC50 and Ki values for promising compounds

  • Analyze inhibition mechanisms (competitive, noncompetitive, uncompetitive)

  • Assess specificity by testing against human SHMT and other PLP-dependent enzymes

  • Evaluate antimicrobial activity against K. pneumoniae clinical isolates

The dual targeting of both the primary SHMT activity and the potential moonlighting alanine racemase function could provide distinct advantages for antimicrobial development, potentially addressing both metabolic and structural aspects of bacterial physiology.

What genetic engineering approaches can enhance K. pneumoniae glyA for biotechnological applications?

Genetic engineering can significantly expand the utility of K. pneumoniae glyA for various research and biotechnological applications:

• Engineering enhanced stability:

  • Identify and modify surface residues to increase solubility

  • Introduce disulfide bridges to stabilize the tertiary structure

  • Implement consensus design approaches based on multiple SHMT sequences

• Substrate specificity modifications:

  • Target active site residues that interact with substrates

  • Create variants with altered or expanded substrate ranges

  • Engineer specificity for non-natural substrates for biocatalytic applications

• Cofactor modifications:

  • Develop variants with altered cofactor preferences

  • Engineer enzymes that can utilize synthetic cofactor analogs

  • Create cofactor-independent variants for specialized applications

• Protein fusion strategies:

  • Generate bifunctional enzymes by fusing glyA with complementary enzymes

  • Create self-immobilizing variants by fusion with cellulose-binding domains

  • Develop biosensor constructs by fusion with fluorescent proteins

For implementing these approaches, researchers should:

• Use computational design tools to predict beneficial mutations

• Implement directed evolution with appropriate selection systems

• Combine rational design with random mutagenesis for optimal results

• Develop high-throughput screening methods specific to the desired property

These engineered variants can find applications in biocatalysis for the synthesis of chiral amino acids, development of biosensors for metabolite detection, and as research tools for studying one-carbon metabolism.

How can systems biology approaches enhance our understanding of K. pneumoniae glyA function?

Systems biology integrates multiple types of biological data to understand complex systems. For K. pneumoniae glyA, several systems approaches can provide comprehensive insights:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data to map glyA-centered networks

  • Identify condition-specific changes in glyA expression and correlate with metabolic shifts

  • Compare wild-type and glyA-perturbed strains to determine systemic effects

  • Implement temporal profiling to capture dynamic responses

• Genome-scale metabolic modeling:

  • Incorporate glyA reactions into existing K. pneumoniae metabolic models

  • Perform flux balance analysis to predict effects of glyA modulation

  • Identify synthetic lethal interactions that involve glyA

  • Simulate metabolic adaptations to environmental changes and drug treatments

• Network analysis:

  • Construct protein-protein interaction networks centered on glyA

  • Map genetic interactions to identify functional relationships

  • Develop pathway models incorporating glyA-dependent reactions

  • Analyze regulatory networks controlling glyA expression

• Computational simulations:

  • Perform molecular dynamics simulations to understand conformational dynamics

  • Develop kinetic models of pathways involving glyA

  • Use machine learning to predict phenotypes associated with glyA variants

  • Implement comparative genomics to identify conserved glyA-associated gene clusters

These systems approaches can reveal how glyA functions within the broader context of K. pneumoniae physiology and identify potential intervention points for antimicrobial development.

How does K. pneumoniae glyA compare functionally to glyA in other bacterial species?

Comparative analysis of glyA across bacterial species reveals important functional similarities and differences with significant research implications:

Based on search results, we can compare glyA across several bacterial species:

In C. pneumoniae, GlyA serves as a source of D-alanine, partially substituting for the absence of dedicated alanine racemases . This moonlighting function represents an interesting evolutionary adaptation that may be shared by K. pneumoniae glyA.

In T. forsythia, the glyA gene is associated with virulence and has been found at higher frequencies (57.14%) in aggressive periodontitis compared to other forms of the disease . This suggests potential virulence-related functions that might be conserved in K. pneumoniae.

For comparative functional analysis, researchers should:

• Express recombinant glyA from multiple species under identical conditions

• Compare kinetic parameters for both primary and secondary reactions

• Perform complementation studies in appropriate knockout strains

• Analyze structural differences that might account for functional variations

What evolutionary insights can be gained from studying K. pneumoniae glyA?

Evolutionary analysis of glyA can provide valuable insights into bacterial adaptation and metabolic evolution:

The search results indicate that GlyA is conserved across diverse bacterial species including E. coli, Chlamydiaceae, and environmental chlamydiae . This conservation suggests essential functions that have been maintained throughout bacterial evolution.

An interesting evolutionary observation comes from comparing alanine racemase and glyA distribution. While environmental chlamydiae genera (Parachlamydia, Protochlamydia, and Waddlia) harbor both dedicated alanine racemases and glyA, Chlamydiaceae lack alanine racemases but retain glyA . This suggests that glyA's moonlighting alanine racemase activity may have allowed gene loss of dedicated alanine racemases in some lineages, representing an elegant example of enzyme promiscuity driving evolutionary streamlining.

To investigate the evolutionary history of K. pneumoniae glyA, researchers should:

• Perform phylogenetic analysis:

  • Construct phylogenetic trees based on glyA sequences from diverse bacterial species

  • Compare glyA phylogeny with species phylogeny to identify potential horizontal gene transfer events

  • Analyze selective pressures using dN/dS ratios to identify regions under purifying or positive selection

• Ancestral sequence reconstruction:

  • Infer ancestral glyA sequences to understand the evolutionary trajectory

  • Express reconstructed ancestral enzymes to characterize their properties

  • Determine if moonlighting functions evolved before or after specialization

• Population genetics:

  • Analyze glyA polymorphisms in K. pneumoniae clinical isolates

  • Correlate genetic variations with phenotypic differences

  • Identify signatures of selection in different ecological niches

These evolutionary insights can illuminate how metabolic enzymes evolve new functions while maintaining their primary activities, with potential implications for understanding bacterial adaptation and pathogenesis.

How can researchers overcome expression and purification challenges with K. pneumoniae glyA?

Based on the search results describing expression of similar bacterial SHMTs, researchers may encounter several challenges when working with K. pneumoniae glyA:

Common challenge 1: Low expression levels
Solutions:

• Optimize codon usage for the expression host

• Test multiple promoter systems (T7, tac, araBAD)

• Evaluate different expression hosts (BL21(DE3), Rosetta, Arctic Express)

• Adjust induction parameters (inducer concentration, temperature, duration)

• Consider fusion tags that enhance expression (MBP, SUMO)

Common challenge 2: Protein insolubility
Solutions:

• Lower induction temperature (25°C as used for C. pneumoniae GlyA)

• Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

• Include solubility enhancers in growth media (sorbitol, betaine)

• Try periplasmic expression with signal peptides

• Use detergents during extraction (N-lauroylsarcosine at 2% for lysis, 0.1% for washing)

Common challenge 3: Loss of cofactor
Solutions:

• Supplement expression media with PLP (50 μM)

• Include PLP in all purification buffers (50 μM)

• Add reducing agents (2 mM DTT) to prevent oxidation

• Consider dialysis rather than dilution methods for buffer exchanges

Common challenge 4: Proteolytic degradation
Solutions:

• Include protease inhibitor cocktails during purification

• Use protease-deficient host strains

• Maintain samples at 4°C during all purification steps

• Minimize purification duration with optimized protocols

Implementation of these strategies should be systematic, testing one variable at a time and documenting results carefully to develop an optimized protocol for K. pneumoniae glyA.

What quality control measures ensure reliable results when working with recombinant K. pneumoniae glyA?

Rigorous quality control is essential for obtaining reliable and reproducible results with recombinant enzymes. For K. pneumoniae glyA, implement the following comprehensive quality control regimen:

• Purity assessment:

  • SDS-PAGE with densitometry analysis (aim for >95% purity)

  • Western blot using tag-specific antibodies

  • Size exclusion chromatography to verify homogeneity

  • Mass spectrometry to confirm correct protein mass and sequence

• Structural integrity verification:

  • UV-visible spectroscopy to confirm PLP binding (characteristic absorption at 425-435 nm)

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to determine stability and proper folding

  • Dynamic light scattering to detect aggregation

• Functional validation:

  • Specific activity determination under standardized conditions

  • Measure kinetic parameters (Km, kcat) for key substrates

  • Inhibition studies with known inhibitors (e.g., D-cycloserine)

  • Substrate specificity profiling to ensure consistent catalytic behavior

• Storage stability monitoring:

  • Activity retention during storage under various conditions

  • Freeze-thaw stability assessment

  • Long-term stability at different temperatures

  • Effect of various stabilizers (glycerol, sucrose, BSA)

Establish acceptance criteria for each parameter and maintain detailed records for batch-to-batch comparison. For critical experiments, consider testing multiple protein batches to ensure reproducibility of results.

For storage, aliquot the purified enzyme to avoid multiple freeze-thaw cycles, add glycerol (20-25%), flash-freeze in liquid nitrogen, and store at -80°C. For experiments requiring extended room temperature incubations, additives such as BSA (0.1 mg/ml) can enhance stability.