Recombinant Neisseria meningitidis serogroup B Phosphoserine aminotransferase (serC)

What is the function of Phosphoserine aminotransferase (SerC) in Neisseria meningitidis?

SerC in N. meningitidis catalyzes the reversible conversion of 3-phosphohydroxypyruvate to phosphoserine and of 3-hydroxy-2-oxo-4-phosphonooxybutanoate to phosphohydroxythreonine. As a member of the class-V pyridoxal-phosphate-dependent aminotransferase family (SerC subfamily), this enzyme plays a critical role in serine biosynthesis and amino acid metabolism in the pathogen . The enzymatic activity is essential for bacterial growth and survival, making it a potential target for therapeutic intervention in meningococcal disease research.

How is SerC structurally characterized in N. meningitidis serogroup B?

SerC in N. meningitidis serogroup B is a protein of approximately 368 amino acids with a molecular mass of around 41.5 kDa . The enzyme contains specific domains characteristic of aminotransferases, including a pyridoxal phosphate (PLP) binding site. The protein's structure facilitates its function in amino acid metabolism through proper substrate recognition and catalytic activity. For structural analysis, researchers typically employ X-ray crystallography or homology modeling based on related aminotransferases from the same family.

What expression systems are most effective for producing recombinant N. meningitidis SerC?

For recombinant expression of N. meningitidis SerC, E. coli-based expression systems (particularly BL21(DE3) strains) are commonly employed using vectors like pET series that provide strong inducible promoters. Optimal expression conditions typically include:

When designing expression constructs, consider incorporating affinity tags (His6, GST) for easier purification while ensuring the tag doesn't interfere with enzymatic activity through control experiments.

How can researchers accurately assess SerC enzyme kinetics in N. meningitidis serogroup B?

To determine SerC enzyme kinetics, researchers should employ spectrophotometric assays that monitor either substrate consumption or product formation. For phosphoserine aminotransferase activity, a coupled enzyme assay system is commonly used:

• Primary reaction: 3-phosphohydroxypyruvate + glutamate → 3-phosphoserine + α-ketoglutarate

• Coupling reaction: α-ketoglutarate + NADH + H⁺ → glutamate + NAD⁺ (catalyzed by glutamate dehydrogenase)

The decrease in NADH absorbance at 340 nm is monitored to calculate reaction rates. For accurate kinetic parameter determination:

• Maintain enzyme concentration significantly below substrate concentration

• Use a range of substrate concentrations (0.1-10× K​m)

• Control temperature (typically 25°C or 37°C)

• Maintain appropriate pH (7.0-8.0) using phosphate or Tris buffer

Data analysis should include Lineweaver-Burk or non-linear regression analysis to determine K​m, k​cat, and catalytic efficiency (k​cat/K​m) .

What strategies can be employed to investigate potential inhibitors of N. meningitidis SerC?

For inhibitor screening against N. meningitidis SerC, researchers can employ:

• High-throughput screening (HTS) approaches using the coupled enzyme assay system described above

• Structure-based virtual screening using computational docking of compound libraries

• Fragment-based drug discovery focusing on the active site and substrate binding regions

When evaluating potential inhibitors:

For physiological relevance, validate promising inhibitors in bacterial growth assays using both wild-type and SerC-complemented strains to confirm specificity .

How does amino acid sequence variation in SerC correlate with virulence among different N. meningitidis serogroups?

To investigate the relationship between SerC sequence variation and virulence:

• Perform comprehensive sequence alignment of SerC proteins from multiple clinical isolates representing different serogroups (particularly A, B, C, W-135, and Y)

• Identify polymorphic sites and correlate them with virulence phenotypes

• Employ site-directed mutagenesis to introduce specific variations and assess their impact on:

  • Enzyme kinetics (catalytic efficiency)

  • Protein stability (thermal shift assays)

  • Bacterial growth rates

  • Virulence in appropriate infection models

Research has shown that N. meningitidis undergoes extensive recombination events affecting metabolic genes, which may contribute to virulence differences between strains . Approximately 40% of meningococcal core genes, including many metabolic genes, show evidence of recombination . When analyzing SerC variations, researchers should consider both the direct enzymatic consequences and the broader metabolic context within different genetic backgrounds.

How does SerC function relate to capsular polysaccharide synthesis in N. meningitidis serogroup B?

While SerC itself is not directly involved in capsule biosynthesis, its role in amino acid metabolism may indirectly influence capsular polysaccharide production. The capsular polysaccharide of serogroup B consists of α2→8-linked polysialic acid, and its synthesis pathway intersects with central metabolism.

Researchers investigating these connections should:

• Employ metabolic flux analysis using isotope-labeled precursors to trace carbon flow between amino acid metabolism and capsule synthesis

• Create controlled SerC expression systems (using inducible promoters) to examine how varying SerC activity levels affect capsule production

• Perform comparative transcriptomics of wild-type vs. SerC-attenuated strains to identify regulatory connections

The capsule is a critical virulence determinant in N. meningitidis, with the cps cluster consisting of 6 regions (D-A-C-E-D′-B) required for biosynthesis, transport, and translocation . Understanding how core metabolism interfaces with capsule production could reveal new therapeutic approaches.

What is the evolutionary significance of SerC conservation across different Neisseria species and serogroups?

SerC is generally well-conserved across Neisseria species as it plays a fundamental role in metabolism. To investigate its evolutionary significance:

• Perform phylogenetic analysis of SerC sequences across:

  • Different Neisseria species (pathogenic and commensal)

  • Various N. meningitidis serogroups

  • Temporally and geographically diverse isolates

• Calculate selection pressures (dN/dS ratios) across the protein sequence to identify:

  • Regions under purifying selection (likely functional domains)

  • Regions under positive selection (potential adaptation sites)

• Correlate SerC sequence variations with recombination events that are known to be prevalent in N. meningitidis

What are the most effective purification strategies for obtaining high-yield, active recombinant N. meningitidis SerC?

For optimal purification of recombinant N. meningitidis SerC:

To maintain enzymatic activity:

• Include pyridoxal-5'-phosphate (PLP, 10-50 μM) in all buffers

• Avoid freeze-thaw cycles; store at -80°C in small aliquots with 10-20% glycerol

• For long-term storage, evaluate protein stability by regular activity measurements

Typical yields from optimized E. coli expression systems should reach 10-20 mg of pure protein per liter of culture .

How can researchers effectively design site-directed mutagenesis experiments to understand structure-function relationships in N. meningitidis SerC?

For structure-function studies of N. meningitidis SerC:

• Prioritize residues for mutagenesis based on:

  • Sequence conservation analysis across the aminotransferase family

  • Structural modeling identifying catalytic and substrate-binding residues

  • Comparison with characterized SerC enzymes from other organisms

• Design mutations strategically:

  • Conservative substitutions (e.g., Asp→Glu) to assess charge requirements

  • Non-conservative substitutions (e.g., Asp→Ala) to eliminate functional groups

  • Cysteine substitutions for subsequent chemical modification studies

• Perform comprehensive functional characterization:

  • Kinetic parameters (Km, kcat) for both forward and reverse reactions

  • Substrate specificity profiles using structurally related compounds

  • Thermal stability assessments using differential scanning fluorimetry

  • Structural verification using circular dichroism or crystallography

This approach has been successfully applied in studies of other aminotransferases and provides insights into catalytic mechanisms and substrate specificity determinants .

What methods are most appropriate for analyzing the immunological properties of recombinant N. meningitidis serogroup B SerC?

For immunological characterization of SerC:

• Assess cellular localization and accessibility:

  • Subcellular fractionation combined with Western blotting

  • Flow cytometry of intact bacteria using anti-SerC antibodies

  • Immunoelectron microscopy for precise localization

• Evaluate immunogenicity:

  • Production of polyclonal antibodies in animal models

  • Epitope mapping using peptide arrays or hydrogen-deuterium exchange mass spectrometry

  • T-cell response analysis using purified recombinant SerC

• Determine cross-reactivity with human proteins:

  • Sequence and structural comparison with human homologs

  • Serum reactivity testing from healthy donors and meningococcal disease patients

  • Inhibition ELISA to assess antibody specificity

These approaches can determine whether SerC represents a potential vaccine antigen or diagnostic marker, which is particularly relevant given the ongoing challenges in developing effective vaccines against N. meningitidis serogroup B due to the similarity of its capsular polysaccharide to human neural cell adhesion molecules .

How does SerC activity in N. meningitidis serogroup B compare to other meningococcal serogroups during host infection?

To compare SerC activity across different serogroups during infection:

• Develop SerC-specific activity assays applicable to complex biological samples

• Perform comparative transcriptomics and proteomics of different serogroups during:

  • In vitro growth in host-mimicking conditions

  • Ex vivo models (human nasopharyngeal tissue, blood)

  • In vivo infection models

• Create reporter strains with SerC promoter fusions to monitor expression dynamics

Research has shown that different N. meningitidis serogroups exhibit distinct metabolic adaptations during infection. Serogroup B strains may have unique metabolic requirements related to their polysialic acid capsule, which differs from the capsular composition of other serogroups like A, C, W-135, and Y . These differences potentially influence SerC expression and activity levels during pathogenesis.

What role might SerC play in the recombination events that contribute to N. meningitidis virulence evolution?

While SerC itself is not directly involved in recombination, researchers can explore:

• Whether SerC sequence diversity correlates with recombination hotspots in the N. meningitidis genome

• If metabolic stress (e.g., serine limitation) influences recombination rates

• How SerC function impacts the fitness of recombinant strains with altered capsular types

N. meningitidis undergoes extensive recombination, with studies identifying 4,026 recombination events and 21 recombinant SNPs for every point mutation within certain clonal complexes . Approximately 40% of meningococcal core genes show evidence of recombination, primarily within metabolic genes and genes involved in DNA replication and repair . As a metabolic enzyme, SerC may influence the selective pressures driving these recombination events.

How can systems biology approaches integrate SerC function into broader metabolic models of N. meningitidis pathogenesis?

To integrate SerC into systems-level understanding:

• Develop constraint-based metabolic models (e.g., Flux Balance Analysis) that incorporate:

  • SerC reaction kinetics and regulation

  • Growth phase-dependent metabolic shifts

  • Host-pathogen metabolic interactions

• Apply multi-scale modeling to connect:

  • Molecular-level enzyme function

  • Cellular-level metabolic networks

  • Population-level infection dynamics

• Use perturbation experiments to validate models:

  • Controlled SerC expression modulation

  • Metabolic precursor supplementation

  • Environmental stress responses

Research has indicated that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence . SerC, as part of central amino acid metabolism, likely plays a role in these metabolic adaptations that contribute to pathogenesis. Systems biology approaches can reveal how seemingly subtle changes in metabolic enzyme function may have far-reaching effects on bacterial fitness and virulence through complex regulatory networks and metabolic interdependencies.