Recombinant Neisseria meningitidis serogroup B Agmatinase (speB)

What is the biochemical function of bacterial agmatinase and how does it relate to N. meningitidis metabolism?

Agmatinase (speB) catalyzes the hydrolysis of agmatine to putrescine and urea, representing a critical step in polyamine biosynthesis. This enzymatic reaction serves as an alternative pathway for polyamine synthesis and plays an important role in regulating agmatine concentrations in bacterial systems . In bacterial species like E. coli, agmatinase functions as part of a metabolic pathway that constitutes the primary mechanism for polyamine synthesis from arginine .

While the specific metabolic role in N. meningitidis requires further investigation, bacterial agmatinases broadly participate in nitrogen metabolism and polyamine production, which are essential for various cellular processes including growth, stress resistance, and potentially virulence. Recent research has revealed that the human enzyme annotated as agmatinase actually hydrolyzes a range of linear guanidino acids rather than agmatine specifically, suggesting that substrate specificity should be thoroughly examined in bacterial homologs as well .

How is the structure of bacterial agmatinase characterized, and what might this tell us about N. meningitidis speB?

The high-resolution X-ray crystal structure of E. coli agmatinase (speB) has revealed that the enzyme adopts a hexameric quaternary structure with 18 chains corresponding to three complete hexamers in the asymmetric unit . Each protomer displays the conserved fold characteristic of the agmatine ureohydrolase family, with remarkably high structural similarity between individual chains .

The active site contains two distinct manganese ions coordinated by highly conserved aspartate and histidine residues that are characteristic of the arginase superfamily . Specifically, in E. coli speB, acidic residues D153 and E274 play crucial roles in catalysis, positioning a hydroxide ion for nucleophilic attack on the substrate . Based on homology to E. coli speB, N. meningitidis agmatinase likely shares similar structural elements, including the metal-binding residues and catalytic machinery, though species-specific variations might influence substrate specificity or regulation.

What expression systems have proven most effective for producing recombinant bacterial agmatinases with proper folding and activity?

Based on successful studies with E. coli speB and other members of the ureohydrolase family, heterologous expression in E. coli remains the predominant system for recombinant production of bacterial agmatinases . When designing expression constructs for N. meningitidis agmatinase, researchers should consider:

• Codon optimization for the expression host

• Inclusion of appropriate affinity tags (typically N-terminal His6) for purification

• Evaluation of multiple E. coli expression strains (BL21(DE3), Rosetta, Arctic Express)

• Testing various induction conditions (temperature, IPTG concentration, duration)

For enzymes requiring metal cofactors like agmatinase, supplementation of the growth medium with manganese (typically 1-5 mM MnCl2) during expression or later during purification is often critical for obtaining catalytically active protein. Expression at lower temperatures (16-18°C) following induction may enhance proper folding of this complex oligomeric protein.

What purification strategy would you recommend for isolating high-purity recombinant N. meningitidis agmatinase while maintaining enzymatic activity?

A multi-step purification protocol leveraging the metal-binding properties of agmatinase would be most appropriate:

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA or Co-NTA resins for His-tagged constructs as the initial capture step

• Ion exchange chromatography (typically anion exchange) to remove contaminating proteins

• Size exclusion chromatography to separate properly folded hexamers from aggregates or incomplete assemblies

• Throughout purification, maintain buffer conditions containing:

  • 20-50 mM Tris or HEPES buffer (pH 7.5-8.0)

  • 100-300 mM NaCl for stability

  • 1-5 mM MnCl2 to ensure metal cofactor availability

  • 1-5 mM DTT or TCEP to prevent oxidation of cysteine residues

  • 5-10% glycerol to improve protein stability

Activity assays should be performed after each purification step to monitor retention of enzymatic function, particularly following any refolding or metal reconstitution procedures. When designing storage conditions, researchers should test stability at various protein concentrations (0.5-5 mg/mL) and consider flash-freezing aliquots in liquid nitrogen with cryoprotectants to preserve long-term activity.

What methods are most reliable for measuring N. meningitidis agmatinase activity, and what controls should be included?

A robust enzymatic assay for N. meningitidis agmatinase should quantify either the consumption of substrate or production of products. Based on the catalytic function of agmatinase, recommended approaches include:

Primary assay methods:

• Spectrophotometric detection of urea formation using diacetyl monoxime or urease-coupled assays

• Fluorometric detection of putrescine using o-phthalaldehyde derivatization

• HPLC separation and quantification of agmatine and putrescine

• LC-MS/MS analysis for highest sensitivity and specificity

Essential controls:

• No-enzyme controls to establish baseline readings

• Heat-inactivated enzyme controls to confirm enzymatic nature of the reaction

• EDTA-treated enzyme to verify metal dependence

• Complementation with Mn2+ to rescue activity after EDTA treatment

• Testing with a range of guanidino acid substrates, not just agmatine, given recent findings with human agmatinase

When testing kinetic parameters, substrate concentration ranges should span at least 0.2-5× Km values, and time-course measurements should establish linear reaction rates. Recent research suggests that agmatinase might hydrolyze a range of linear guanidino acids, so substrate specificity determination should be a priority for N. meningitidis agmatinase characterization .

How can site-directed mutagenesis be used to elucidate the catalytic mechanism of N. meningitidis agmatinase?

Based on structural and functional studies of E. coli speB, a systematic mutagenesis approach should target:

• Metal-binding residues: Mutation of conserved histidine and aspartate residues that coordinate the manganese ions in the active site will directly test their role in catalysis

• Catalytic residues: Acidic residues equivalent to E. coli speB's D153 and E274 that likely position the nucleophilic hydroxide for attack on the substrate

• Substrate-binding pocket residues: Amino acids that form the binding pocket can be mutated to assess their contribution to substrate specificity, particularly focusing on residues that might interact with the guanidino group

• Oligomerization interface residues: Mutations disrupting the hexameric assembly can reveal whether the quaternary structure is essential for catalytic activity

For each mutant, comprehensive characterization should include:

• Thermal stability assessment (DSF/DSC)

• Metal content analysis (ICP-MS)

• Steady-state kinetic parameters (kcat, Km)

• pH dependence profiles

• Substrate specificity determination

This systematic approach will provide a comprehensive understanding of structure-function relationships in N. meningitidis agmatinase and could reveal unique features compared to homologs from other species.

What evidence exists for agmatinase's role in N. meningitidis virulence or persistence, and how could this be further investigated?

• Protection against oxidative stress during host immune responses

• Biofilm formation and colonization

• Resistance to host antimicrobial peptides

• Regulation of gene expression during infection

To investigate the role of agmatinase in N. meningitidis virulence, researchers should consider:

Genetic approaches:

• Construction of agmatinase knockout strains

• Complementation studies with wild-type and catalytically inactive mutants

• Conditional expression systems to control agmatinase levels

Phenotypic assays:

• Growth curves under various stress conditions

• Biofilm formation capacity

• Adhesion to and invasion of human cell lines

• Serum resistance assays

• Mouse infection models comparing wild-type and mutant strains

Transcriptomic/proteomic analyses:

• RNA-seq to identify genes differentially expressed in agmatinase mutants

• Proteomics to detect changes in protein abundance

• Metabolomics focusing on polyamine pathway intermediates

These comprehensive approaches would provide insights into whether agmatinase represents a potential therapeutic target for meningococcal infections.

Could N. meningitidis agmatinase be incorporated into vaccine development strategies, and what experimental evidence would support this approach?

For N. meningitidis agmatinase to be considered a viable vaccine candidate, several criteria would need to be satisfied:

• Surface accessibility: While traditional bacterial enzymes are often cytoplasmic, evidence of surface exposure or secretion would significantly enhance vaccine potential

• Conservation across strains: Analysis of sequence conservation across diverse clinical isolates would determine if agmatinase could provide broad protection

• Immunogenicity: Studies would need to demonstrate that recombinant agmatinase elicits robust antibody responses in animal models

• Functional antibodies: Crucial evidence would include demonstration that anti-agmatinase antibodies exhibit bactericidal activity in serum bactericidal assays (SBA), which are considered correlates of protection against meningococcal disease

Current outer membrane vesicle (OMV) vaccines against N. meningitidis serogroup B incorporate multiple antigens . If agmatinase shows promise, it could potentially be included in next-generation vaccines either as a purified recombinant protein or by enhancing its expression in engineered OMVs.

Notably, the study of N. cinerea OMVs as vaccine candidates demonstrated that sera from immunized mice displayed high bactericidal activity against N. meningitidis strains expressing the targeted antigens, with titers as high as 32,768 against certain strains . Similar approaches could be used to evaluate agmatinase-containing vaccine formulations.

What structural biology techniques would be most informative for characterizing N. meningitidis agmatinase interactions with substrates and inhibitors?

A multi-technique structural biology approach would provide comprehensive insights into N. meningitidis agmatinase:

X-ray crystallography:

• Apo-enzyme structure determination at high resolution (≤2.0 Å)

• Co-crystallization with substrates, substrate analogs, and potential inhibitors

• Metal-depleted structures to understand conformational changes

Cryo-electron microscopy:

• Particularly valuable if crystallization proves challenging

• Could reveal dynamic aspects of the hexameric assembly

Small-angle X-ray scattering (SAXS):

• Characterization of oligomeric state and conformational changes in solution

• Complementary to crystallographic data

NMR spectroscopy:

• Focused on substrate binding and dynamics

• 15N-HSQC experiments to monitor chemical shift perturbations upon ligand binding

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

• Mapping regions with altered solvent accessibility upon substrate binding

• Identifying conformational changes not captured in static crystal structures

The E. coli speB structure determined through X-ray crystallography revealed important insights about its hexameric assembly and active site architecture . Similar studies with N. meningitidis agmatinase would form the foundation for structure-guided inhibitor design targeting this enzyme.

How might naturally occurring variants of N. meningitidis agmatinase differ in substrate specificity and catalytic efficiency?

Recent findings with human agmatinase revealed that naturally occurring variants exhibit different substrate preferences . This suggests that N. meningitidis agmatinase variants might similarly display altered catalytic properties. To investigate this:

• Sequence analysis across strains:

  • Identify polymorphic positions in clinical isolates

  • Focus on residues within the substrate-binding pocket

  • Perform evolutionary analysis to detect positions under selective pressure

• Biochemical characterization of variants:

  • Express and purify recombinant versions of identified natural variants

  • Compare substrate specificity profiles using a panel of potential substrates

  • Determine full kinetic parameters (kcat, Km) for each variant with each substrate

  • Assess temperature and pH optima for activity

• Structural analysis:

  • Obtain crystal structures of key variants

  • Perform molecular dynamics simulations to understand how amino acid substitutions affect substrate binding and catalysis

The study on human enzyme indicates that "a negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis" . This structural requirement could be assessed in N. meningitidis agmatinase variants to determine if similar substrate recognition mechanisms operate.

How can metabolomics approaches be integrated with genetic manipulation to understand the physiological role of agmatinase in N. meningitidis?

An integrated systems biology approach combining metabolomics with genetic manipulation would provide comprehensive insights into agmatinase function:

Experimental design:

• Strain construction:

  • Agmatinase knockout strain (ΔspeB)

  • Complemented strain (ΔspeB+speB)

  • Catalytically inactive mutant (ΔspeB+speBmut)

  • Overexpression strain (speB↑)

• Metabolomic profiling:

  • Targeted LC-MS/MS analysis focusing on polyamines and related metabolites

  • Untargeted metabolomics to identify unexpected metabolic perturbations

  • Isotope labeling with 13C-arginine to trace metabolic flux through the agmatine pathway

  • Time-course analysis during different growth phases and stress conditions

• Multi-omics integration:

  • Parallel transcriptomic analysis to identify compensatory changes in gene expression

  • Correlation of metabolite levels with protein abundance

  • Network analysis to identify perturbed pathways

This approach would reveal not only the direct metabolic consequences of agmatinase activity but also broader effects on bacterial physiology and potential metabolic adaptations that occur when the enzyme is absent or dysfunctional.

What are the implications of recent findings questioning the substrate specificity of agmatinase for future research on N. meningitidis speB?

The recent discovery that human agmatinase (AGMAT) actually hydrolyzes a range of linear guanidino acids rather than agmatine specifically has profound implications for research on bacterial agmatinases . For N. meningitidis agmatinase research, this finding necessitates:

• Comprehensive substrate screening:

  • Testing activity with various guanidino acids beyond agmatine

  • Determining structure-activity relationships for substrate recognition

  • Investigating whether substrate preference varies across different N. meningitidis strains

• Metabolic pathway reassessment:

  • Identifying which guanidino acids are present in N. meningitidis under various conditions

  • Determining if the true physiological substrates differ from agmatine

  • Exploring potential novel metabolic pathways involving these alternative substrates

• Nomenclature and annotation considerations:

  • If substrate specificity differs substantially from traditional agmatinase activity, researchers may need to consider renaming the enzyme, similar to the proposed renaming of human AGMAT to guanidino acid hydrolase (GDAH)

  • Structural comparison with human GDAH could reveal whether the substrate preference mechanisms are conserved

• Evolutionary perspective:

  • Comparative analysis across bacterial species to understand how substrate specificity evolved

  • Assessment of whether substrate preference correlates with ecological niche or pathogenicity

This paradigm shift in understanding substrate specificity could lead to discoveries of previously unrecognized metabolic pathways in N. meningitidis and potentially identify new targets for therapeutic intervention.